Inhibition of TREM receptor signaling with peptide variants

ABSTRACT

Peptides are provided consisting of L- and/or D-amino acids and combinations thereof, which affect myeloid cells by action on the triggering receptors expressed on myeloid cells (TREMs), including TREM-1 and TREM-2. The peptides act on the TREM/DAP-12 signaling complex. Also provided are lipid and sugar conjugated peptides comprising L- or D-amino acids. A method is provided of designing the peptides and lipid- and/or sugar-conjugated peptides comprising L- or D-amino acids. The disclosure relates to the therapy of various myeloid cell-related disease states involving the use of these peptides and compounds. The peptides and compounds are useful in the treatment and/or prevention of a disease or condition where myeloid cells are involved or recruited. The peptides of the present invention also are useful in the production of medical devices comprising peptide matrices (for example, medical implants and implantable devices).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation patent application claiming priority toand the benefit of a co-pending U.S. application Ser. No. 13/501,992filed on Apr. 13, 2012, which is a National Stage entry of InternationalApplication No. PCT/US10/52566, filed on Oct. 13, 2010, which claimspriority to and the benefit of U.S. provisional application Ser. No.61/251,283, filed Oct. 13, 2009. The entire content of theaforementioned applications is incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted Sequence Listing with a file named“SBK-003CON_Sequences_ST25.txt”, created on Feb. 8, 2014, and having asize of 3 kilobyte. The sequence listing contained in this ASCIIformatted document is part of the specification and is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to peptides and compounds which affect theactivating TREM receptor signaling pathway in granulocytes, monocytes,macrophages, neutrophils, microglia, dendritic cells, osteoclasts,platelets and other myeloid cells. The present invention further relatesto the treatment or prevention of cancer, allergic diseases,inflammatory bowel disease, empyema, acute mesenteric ischemia,hemorrhagic shock, autoimmune diseases, including but not limited to,rheumatoid arthritis and other rheumatic diseases, sepsis and otherinflammatory or other condition involving myeloid cell activation, and,more particularly, TREM receptor-mediated cell activation. In oneembodiment, TREM-1/DAP-12 receptor complex signaling is inhibited byvariant peptides binding to the transmembrane region of the DAP-12subunit.

BACKGROUND OF THE INVENTION

1. TREM Receptors

Innate immunity is crucial for host survival during the early stages ofinfection. However, fine-tuning of this response is absolutely crucialto prevent excessive inflammation and tissue damage (Ford J. W. &McVicar D. W. Curr Opin Immunol 2009; 21, 38-46). Pathogen sensing isachieved through a constellation of pathogen recognition receptors, suchas the toll-like receptors (TLR), which activate innate immune cells toclear the pathogen and to shape the adaptive immune response. Otherinnate immune receptors, such as the triggering receptor expressed onmyeloid cells (TREM), modulate the innate response either by amplifyingor dampening TLR-induced signals, and thus play crucial roles infine-tuning of the inflammatory response. Since the discovery oftriggering receptor expressed on myeloid cells (TREM)-1 in 2000,evidence documenting the profound ability of the TREM and TREM-likereceptors to regulate inflammation has rapidly accumulated (Bouchon etal. J Immunol 2000; 164:4991-5; Ford J. W. & McVicar D. W. Curr OpinImmunol 2009; 21, 38-46; Bouchon et al. Nature 2001; 410:1103-7; GibotS. Crit Care 2005; 9:485-9; Gibot et al. J Exp Med 2004; 200:1419-26;Gibot et al. Shock 2009; 32:633-7; Gibot et al. Crit Care Med 2008;36:504-10; Klesney-Tait et al. Nat Immunol 2006; 7:1266-73; Murakami etal. Arthritis Rheum 2009; 60:1615-23; Sharif 0. & Knapp S. Immunobiology2008; 213:701-13; Ling et al. Chinese Med J 2010; 123:1561-5). The TREMand TREM-like receptors are a structurally related protein familyencoded by genes clustered on mouse chromosome 17C3 and human chromosome6p21. The TREM cluster includes genes encoding TREM-1, TREM-2 and, inthe mouse, TREM-3, as well as the ‘TREM-like’ genes. The ‘TREM-like’genes Treml1 and Treml2 in mouse, and TREML1 and TREML2 in humans,encode TREM-like transcripts 1 and 2 (TLT-1 and TLT-2, respectively).Monocytes, macrophages, myeloid dendritic cells, plasmacytoid dendriticcells, neutrophils, microglia, osteoclasts, and platelets all express atleast one member of the TREM family, underscoring the importance ofthese proteins in the regulation of innate resistance. are expressed ona variety of innate cells of the myeloid lineage including neutrophils,monocytes, macrophages, microglia, osteoclasts, and dendritic cells, aswell as on megakaryocytes and platelets.

Recent work on the TREM family includes: characterization of a newreceptor expressed on plasmacytoid dendritic cells; definition of a keyrole for TREM in sepsis, cancer, inflammatory bowel disease and multiplesclerosis; an expanded list of diseases associated with the release ofsoluble forms of TREM proteins; and identification of the first wellcharacterized TREM ligand: B7-H3, a ligand for TLT-2. Moreover, analysisof TREM signaling has now identified key regulatory components anddefined pathways that may be responsible for the complex functionalinteractions between the TREM and TLRs. Together these findings definethe TREM receptors as pluripotent modifiers of disease through theintegration of inflammatory signals with those associated with leukocyteadhesion.

2. TREM-Related Pathologies

2.1. Sepsis

Septicemia, an invasion of the bloodstream by virulent bacteria thatmultiply and discharge their toxic products, is the serious andsometimes fatal disorder, commonly known as blood poisoning. Theinvasive organisms are usually streptococci or staphylococci but may beany type of bacteria. Septicemia is an extremely dangerous disorderbecause it spreads rapidly throughout the body. If bacteria continue tomultiply in the bloodstream and the condition progresses to septicshock, blood pressure plummets and organ systems begin to shut down.Septic shock is characterized by massive release of proinflammatorymediators and leads not only to tissue damage, but also to haemodynamicchanges, multiple organ failure (multiple-organ dysfunction syndrome,MODS), and ultimately death. More than 750,000 cases of sepsis occurannually in the US, and 215,000 of those afflicted die even withintensive medical care that includes antibiotics, intravenous fluids,blood transfusions, kidney dialysis, nutritional and respiratory supportand sometimes surgery to remove the source of an infection. Theincidence of sepsis has nearly doubled in the last decade and isexpected to rise further, as the population ages and more people survivewith conditions that leave them vulnerable. Despite the use of potentantibiotics and advanced resuscitative equipment costing $17 billion ayear, septic shock remains the most common cause of death innon-coronary intensive care units (ICUs). Despite advances in methodsand compositions for treatment of sepsis (US Pat Appl 20090012025),activated protein C (Xigris, drotrecogin-alpha, marketed by Eli Lilly)is the only FDA-approved drug for sepsis. However, Xigris has a verylimited use—only in patients with high risk of death. Xigris should onlybe administered in an ICU and has significantly less effectiveprotection if delayed in practice. Also, its use is limited tonon-surgical patients due to the adverse effects on coagulation. Thus,there is a great need for an effective novel treatment for sepsis.

Initial findings established TREM-1 as an amplifier of the systemicinflammatory response syndrome associated with sepsis. Blockade ofTREM-1 has been shown to protect mice against lipopolysaccaride(LPS)-induced shock, as well as microbial sepsis caused by liveEscherichia coli or caecal ligation and puncture (Bouchon et al. Nature2001; 410, 1103-7). These results demonstrate a critical function ofTREM-1 in acute inflammatory responses to bacteria and implicate TREM-1as a promising therapeutic target for sepsis.

2.2. Acute Mesenteric Ischemia

Acute mesenteric ischemia is an abdominal emergency associated with 60to 90% mortality (Lock G. Best Pract Res Clin Gastroenterol 2001;15:83-98; Gibot et al. Crit Care Med 2008; 36:504-10). Although ischemiaby itself induces little damage, reperfusion of the previously ischemicorgan can yield to remote organ injury and life-threatening multipleorgan failure. Even though numerous modalities and substances have beenstudied to reduce gut ischemia/reperfusion (I/R)-induced mortality, nonehave been entirely successful. As such, the development of effectivestrategies for preventing and treating circulatory collapse and organinjury after gut I/R is critical for the improvement of patient outcomeunder such conditions.

Recently, inhibition of TREM-1 has been shown to prevent an I/R-inducedmarked increase in ileal mucosal permeability and an associatedbacterial translocation and to delay mortality (Gibot et al. Crit CareMed 2008; 36:504-10), indicating that the inhibition of the TREM-1pathway may be useful during acute mesenteric ischemia.

2.3. Hemorrhagic Shock

Hemorrhagic shock is a condition of reduced tissue perfusion, resultingin the inadequate delivery of oxygen and nutrients that are necessaryfor cellular function. Whenever cellular oxygen demand outweighs supply,both the cell and the organism are in a state of shock. Hemorrhagicshock is primarily caused by traumatic injury, from automobileaccidents, bullet or knife wounds, and falls. Trauma causesapproximately 150,000 deaths per year, and is the leading cause of deathin the population under age 45 in the United States. The resulting lossof productive life years exceeds that of any other disease, withestimated societal costs of >$450 billion annually. Most trauma deathsresult from insufficient tissue perfusion due to excessive blood loss.Clinical management of hemorrhagic shock relies on massive and rapidinfusion of fluids to maintain blood pressure. However, the majority ofvictims with severe blood loss do not respond well to fluid restoration.The development of effective strategies for resuscitation of traumaticblood loss, therefore, is urgently needed.

Recently, it has been shown that early inhibition of the TREM-1 pathwaymay be useful during severe hemorrhagic shock in rats in preventingorgan dysfunction and improving survival (Gibot et al. Shock 2009;32:633-7).

2.4. Rheumatoid Arthritis and Other Rheumatic Diseases

Rheumatic diseases are characterized by inflammation (signs are rednessand/or heat, swelling, and pain) and loss of function of one or moreconnecting or supporting structures of the body. They especially affectjoints, tendons, ligaments, bones, and muscles. Common symptoms arepain, swelling, and stiffness. Some rheumatic diseases can also involveinternal organs. There are more than 100 rheumatic diseases includingbut not limiting to arthritis, ankylosing spondylitis, fibromyalgia,lupus, scleroderma, polymyositis, dermatomyositis, polymyalgiarheumatica, bursitis, tendinitis, vasculitis, carpal tunnel syndrome,complex regional pain syndrome, juvenile arthritis, Lyme disease,systemic lupus erythematosus, Kawasaki disease, fibromyalgia, andchronic fatigue syndrome. Rheumatic diseases may cause pain, stiffness,and swelling in the joints and other supporting body structures, such asmuscles, tendons, ligaments, and bones. However, rheumatic diseases canaffect other areas of the body, including internal organs. Somerheumatic diseases involve connective tissues (called connective tissuediseases), while others may be caused by autoimmune disorders, which arediseases involving the body's immune system attacking its own healthycells and tissues. Rheumatic diseases are the leading cause ofdisability among persons age 65 and older. According to the Centers forDisease Control and Prevention, as of 2002, more than 70 million peoplein the U.S. have some form of arthritis (one in every three adults).This includes roughly 300,000 children that suffer from some form ofarthritis or rheumatic disease, and millions more are at risk ofdeveloping one of these diseases. Most persons over the age of 75 areaffected with osteoarthritis (also called degenerative joint disease) inat least one joint, making this condition a leading cause of disabilityin the US. Rheumatoid arthritis (RA) is an autoimmune diseasecharacterized by synovial hyperplasia with massive infiltration ofinflammatory cells, which leads to degeneration of cartilage, erosion ofbone, and ultimately loss of function in the affected joints. Rheumatoidarthritis is the most crippling form of arthritis and affectsapproximately 2.1 million Americans and two to three times more womenthan men. Further, the average onset for rheumatoid arthritis is betweenthe ages of 20 and 45 years old. Currently, arthritis disables 19million Americans and takes a $128 billion toll annually on the U.S.economy in direct and indirect medical costs.

Recently, blockade of TREM-1 has been shown to represent a new promisingapproach to rheumatic diseases that is safer than the presentlyavailable immunosuppressive treatments (Murakami et al. Arthritis Rheum2009; 60:1615-23).

2.5. Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) accounts for about 87% of all lungcancer patients and affects more than 1.2 million people a year witharound 1.1 million deaths annually in the US and worldwide. Estimatednew cases and deaths from lung cancer (non-small cell and small cellcombined) in the United States in 2010 are 222,520 and 157,300,respectively. Despite advances made in cytotoxic chemotherapy, NSCLCstill kills more patients than breast, colon and prostate cancer takentogether. Thus, there is a great need for an effective novel treatmentfor NSCLC. However, although lung cancer, and particularly primaryNSCLC, is the leading cause of malignancy-related mortality in theUnited States, the biology of this devastating disease is complex andpoorly understood.

Recent findings established that TREM-1 and the inflammatory responseplay an important role in cancer progression. It has been shown thatcancer cells can directly up-regulate TREM-1 expression in patients'macrophages and that TREM-1 expression in tumor-associated macrophagesis associated with cancer recurrence and poor survival of patients withNSCLC (Ho et al. Am J Respir Crit Care Med 2008; 177:763-70). Theseresults demonstrate a critical function of TREM-1 in cancer progressionand implicate TREM-1 as a promising target for the development of newrational anticancer therapy. It can be expected that blocking activationof the TREM-1 pathway may significantly improve survival of patientswith NSCLC.

3. Current Approaches to Inhibit the TREM-1 Pathway

Antibodies have been considered as clinically significant therapeuticagents for various TREM-related pathologies (US Pat Appl 20080247955;Piccio et al. Eur J Immunol 2007; 37:1290-301). However, antibodytherapy poses serious disadvantages. First, as antibodies are naturalproducts they must be produced in cell lines or other live expressionsystems. This raises a question that there could be contamination ofantibody preparations by infectious agents such as prions or viruses.Although tight regulation and regulatory vigilance and surveillance canreduce this concern, the need for ongoing monitoring and testing forcontamination contributes to the high cost of developing andadministering antibody therapies. In addition, antibody-based therapiesrequire considerable logistical support. As antibodies are proteins,they cannot be given orally, except for those used to treat certaintypes of mucosal infectious diseases, and therefore, systemicadministration is required. Another serious disadvantage ofantibody-based therapies is the high costs of production, storage, andadministration. Moreover, long infusions (i.e., for example, an hour orlonger) require a hospital environment and are often associated withmild to very severe side effects. For example, in one trial, in whichfour patients in the U.K. were given an anticancer antibody reactiveagainst an important receptor on T cells (CD28) severe andlife-threatening responses were observed; the cause is at present notunderstood. This makes large-scale clinical applications of a number ofmonoclonal antibodies with demonstrated therapeutic activity impossibleor, at least, severely compromised. Fast degradation of the administeredantibodies is another drawback of antibody-based therapy.

As described in US Pat Appls 20090081199 and 20030165875, fusionproteins between human IgG1 constant region and the extracellular domainof mouse TREM-1 or that of human TREM-1 can be used, as a “decoy”receptor, to inhibit TREM-1. However, these large protein molecules haveposes serious disadvantages similar to those of antibodies.

U.S. Pat. No. 6,420,526 entitled “186 Secreted Proteins” claimsunspecified and unexemplified isolated fragments of TREM-1 containing atleast 30 contiguous amino acids of human TREM-1. No biological datarelating to such fragments are provided.

Peptides based on TREM-1-derived sequences for disrupting TREM-1function presumably by blocking binding of the receptor with its cognateligand have also been disclosed (US Pat Appl 20060246082) or published(Murakami et al. Arthritis Rheum 2009; 60:1615-23; Gibot et al. CritCare Med 2008; 36:504-10; Gibot et al. Shock 2009; 32:633-7). Despitemultiple advantages of these peptides as compared to antibodies, theyhave relatively low efficacy in terms of inhibiting TREM-1, thus havinga high potential for toxicity and side effects. For example, thesystemic administration of a synthetic TREM-1 peptide that mimics shorthighly interspecies-conserved extracellular domains of TREM-1 and isoften called as LP-17 suppresses collagen-induced arthritis, althoughthe effect is not as complete as that observed following viral genetransfer (Murakami et al. Arthritis Rheum 2009; 60:1615-23).

What is needed is a broad-based TREM-targeted therapy designed todisrupt protein-protein interactions that may be administered to treatvarious TREM-related pathologies safely and effectively. It is thereforethe object of the present invention to provide therapeutic compoundsthat can be used to treat various TREM-related disorders.

SUMMARY OF THE INVENTION

The present invention relates to peptides and compounds, which affectmyeloid cells by action on the triggering receptors expressed on myeloidcells (TREMs), including TREM-1 and TREM-2. The present inventionfurther relates to the prevention and therapy of various myeloidcell-related disease states involving the use of these peptides andcompounds. Specifically, the peptides and compounds are useful in thetreatment and/or prevention of a disease or condition where myeloidcells are involved or recruited. The peptides of the present inventionalso are useful in the production of medical devices comprising peptidematrices (for example, medical implants and implantable devices). In oneembodiment, TREM-1 signaling is inhibited by variant TREM-1 peptidesbinding to the transmembrane region of the DAP-12 subunit.

In one embodiment, the present invention contemplates a variant TREM-1peptide inhibitor comprising at least one amino acid addition and/orsubstitution that optimizes binding to a DAP-12 subunit relative to theTREM-1 subunit transmembrane domain (TREM-1 TMD:I-V-I-L-L-A-G-G-F-L-S-K-S-L-V-F-S-V-L-F-A)(SEQ ID NO: 4). In oneembodiment, the peptide further comprises a C-terminal and/or anN-terminal sugar conjugate. In one embodiment, the sugar conjugate is1-amino-glucose succinate. In one embodiment, the peptide furthercomprises a C-terminal and/or an N-terminal lipid conjugate. In oneembodiment, the lipid conjugate is selected from the group comprising2-aminododecanoate or myristoylate. In one embodiment, the lipidconjugate is selected from the group comprising Gly-Tris-monopalmitate,Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment, thepeptide comprises a cyclic peptide. In one embodiment, the peptidecomprises a disulfide-linked dimer. In one embodiment, the peptideinhibitor includes amino acids selected from the group including, butnot limited to, L-amino acids, or D-amino acids.

In another embodiment, the present invention contemplates a methodcomprising: a) providing; i) a patient having at least one symptom of amedical condition or condition where myeloid cells are involved orrecruited; and ii) a variant TREM-1 peptide inhibitor comprising atleast one amino acid addition and/or substitution that optimizes bindingto a DAP-12 subunit relative to the TREM-1 TMD capable of reducing saidTREM-1-mediated cell activation; b) administering said inhibitor to saidpatient under conditions such that said at least one symptom is reduced.In one embodiment, the medical condition comprises sepsis. In oneembodiment, the medical condition comprises non-small cell lung cancer(NSCLC). In one embodiment, the medical condition comprises inflammatorybowel disease. In one embodiment, the medical condition comprises acutemesenteric ischemia. In one embodiment, the medical condition compriseshemorrhagic shock. In one embodiment, the medical condition comprises arheumatic disease. In one embodiment, the rheumatic disease is selectedfrom the group consisting of arthritis, ankylosing spondylitis,fibromyalgia, lupus, scleroderma, polymyositis, dermatomyositis,polymyalgia rheumatica, bursitis, tendinitis, vasculitis, carpal tunnelsyndrome, complex regional pain syndrome, juvenile arthritis, Lymedisease, systemic lupus erythematosus, Kawasaki disease, fibromyalgia,and chronic fatigue syndrome.

In one embodiment, the present invention contemplates a peptideinhibitor comprising an amino acid sequence consisting ofG-X₁-X₂-L-S-X₃-X₄-L-V-X₅-X₆-X₇-X₈-X₉-X₁₀ (SEQ ID NO: 1), wherein X₁consists of G, C or is selected from the group consisting of R, K or H;X₂ is selected from the group consisting of L, F or I; X₃ is selectedfrom the group consisting of R, K or H; X₄ is selected from the groupconsisting of S or T; X₅ consists of F or is selected from the groupconsisting of R, K or H; X₆ consists of S, I, L or nothing; X₇ consistsof V, I, L, G or nothing; and X₈, X₉, and X₁₀ consist of L, F, A ornothing. In one embodiment, the peptide further comprises a C-terminaland/or an N-terminal sugar conjugate. In one embodiment, the sugarconjugate is 1-amino-glucose succinate. In one embodiment, the peptidefurther comprises a C-terminal and/or an N-terminal lipid conjugate. Inone embodiment, the lipid conjugate is selected from the groupcomprising 2-aminododecanoate or myristoylate. In one embodiment, thelipid conjugate is selected from the group comprisingGly-Tris-monopalmitate, Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate.In one embodiment, the peptide comprises a cyclic peptide. In oneembodiment, the peptide comprises a disulfide-linked dimer. In oneembodiment, the peptide inhibitor includes amino acids selected from thegroup including, but not limited to, L-amino acids, or D-amino acids.

In another embodiment, the present invention contemplates a methodcomprising: a) providing; i) a patient having at least one symptom of amedical condition or condition where myeloid cells are involved orrecruited; and ii) a peptide inhibitor comprising an amino acid sequenceconsisting of G-X₁-X₂-L-S-X₃-X₄-L-V-X₅-X₆-X₇-X₈-X₉-X₁₀ (SEQ ID NO: 1),wherein X₁ consists of G, C or is selected from the group consisting ofR, K or H; X₂ is selected from the group consisting of L, F or I; X₃ isselected from the group consisting of R, K or H; X₄ is selected from thegroup consisting of S or T; X₅ consists of F or is selected from thegroup consisting of R, K or H; X₆ consists of S, I, L or nothing; X₇consists of V, I, L, G or nothing; and X₈, X₉, and X₁₀ consist of L, F,A or nothing capable of reducing said TREM-1-mediated cell activation;b) administering said inhibitor to said patient under conditions suchthat said at least one symptom is reduced. In one embodiment, themedical condition comprises sepsis. In one embodiment, the medicalcondition comprises NSCLC. In one embodiment, the medical conditioncomprises inflammatory bowel disease. In one embodiment, the medicalcondition comprises acute mesenteric ischemia. In one embodiment, themedical condition comprises hemorrhagic shock. In one embodiment, themedical condition comprises a rheumatic disease. In one embodiment, therheumatic disease is selected from the group consisting of arthritis,ankylosing spondylitis, fibromyalgia, lupus, scleroderma, polymyositis,dermatomyositis, polymyalgia rheumatica, bursitis, tendinitis,vasculitis, carpal tunnel syndrome, complex regional pain syndrome,juvenile arthritis, Lyme disease, systemic lupus erythematosus, Kawasakidisease, fibromyalgia, and chronic fatigue syndrome.

In one embodiment, the present invention contemplates a peptideinhibitor comprising an amino acid sequence consisting ofX₁-X₂-X₃-G-F-L-S-K-S-L-V-R-V-X₄-X₅ (SEQ ID NO: 2), wherein X₁ consistsof G, C or nothing; and X₂, X₃, X₄, and X₅ consist of K, R, or nothing.In one embodiment, the peptide further comprises a C-terminal and/or anN-terminal sugar conjugate. In one embodiment, the sugar conjugate is1-amino-glucose succinate. In one embodiment, the peptide furthercomprises a C-terminal and/or an N-terminal lipid conjugate. In oneembodiment, the lipid conjugate is selected from the group comprising2-aminododecanoate or myristoylate. In one embodiment, the lipidconjugate is selected from the group comprising Gly-Tris-monopalmitate,Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment, thepeptide comprises a cyclic peptide. In one embodiment, the peptidecomprises a disulfide-linked dimer. In one embodiment, the peptideinhibitor includes amino acids selected from the group including, butnot limited to, L-amino acids, or D-amino acids.

In one embodiment, the present invention contemplates a methodcomprising: a) providing; i) a patient having at least one symptom of amedical condition or condition where myeloid cells are involved orrecruited; and ii) a peptide inhibitor comprising an amino acid sequenceconsisting of X₁-X₂-X₃-G-F-L-S-KS-L-V-R-V-X₄-X₅ (SEQ ID NO: 2), whereinX₁ consists of G, C or nothing; and X₂, X₃, X₄, and X₅ consist of K, R,or nothing capable of reducing said TREM-1-mediated cell activation; b)administering said inhibitor to said patient under conditions such thatsaid at least one symptom is reduced. In one embodiment, the medicalcondition comprises sepsis. In one embodiment, the medical conditioncomprises NSCLC. In one embodiment, the medical condition comprisesinflammatory bowel disease. In one embodiment, the medical conditioncomprises acute mesenteric ischemia. In one embodiment, the medicalcondition comprises hemorrhagic shock. In one embodiment, the medicalcondition comprises a rheumatic disease. In one embodiment, therheumatic disease is selected from the group consisting of arthritis,ankylosing spondylitis, fibromyalgia, lupus, scleroderma, polymyositis,dermatomyositis, polymyalgia rheumatica, bursitis, tendinitis,vasculitis, carpal tunnel syndrome, complex regional pain syndrome,juvenile arthritis, Lyme disease, systemic lupus erythematosus, Kawasakidisease, fibromyalgia, and chronic fatigue syndrome.

In one embodiment, the present invention contemplates a peptideinhibitor comprising an amino acid sequence consisting ofX₁-X₂-X₃-L-X₄-X₅-X₆-X₇-G-X₈-L-S-K-X₉-L-V-F-X₁₀-X₁₁-L-F-X₁₂-X₁₃-X₁₄-X₁₅(SEQ ID NO: 3), wherein X₁ consists of G or nothing; and X₂, X₃, X₁₄,and X₁₅ consist of K, R, or nothing; X₄, X₅, X₆, and X₇ consist of P, A,V, C, L, I, S, G or nothing; X₈ consists of F, L or I; X₉ consists of Sor T; X₁₀, X₁₁, X₁₂, and X₁₃ consist of S, I, L, G, V, A, or nothing. Inone embodiment, the peptide further comprises a C-terminal and/or anN-terminal sugar conjugate. In one embodiment, the sugar conjugate is1-amino-glucose succinate. In one embodiment, the peptide furthercomprises a C-terminal and/or an N-terminal lipid conjugate. In oneembodiment, the lipid conjugate is selected from the group comprising2-aminododecanoate or myristoylate. In one embodiment, the lipidconjugate is selected from the group comprising Gly-Tris-monopalmitate,Gly-Tris-dipalmitate, or Gly-Tris-tripalmitate. In one embodiment, thepeptide comprises a cyclic peptide. In one embodiment, the peptidecomprises a disulfide-linked dimer. In one embodiment, the peptideinhibitor includes amino acids selected from the group including, butnot limited to, L-amino acids, or D-amino acids.

In one embodiment, the present invention contemplates a methodcomprising: a) providing; i) a patient having at least one symptom of amedical condition or condition where myeloid cells are involved orrecruited; and ii) a peptide inhibitor comprising an amino acid sequenceconsisting ofX₁-X₂-X₃-L-X₄-X₅-X₆-X₇-G-X₈-L-S-K-X₉-L-V-F-X₁₀-X₁₁-L-F-X₁₂-X₁₃-X₁₄-X₁₅(SEQ ID NO: 3), wherein X₁ consists of G or nothing; and X₂, X₃, X₁₄,and X₁₅ consist of K, R, or nothing; X₄, X₅, X₆, and X₇ consist of P, A,V, C, L, I, S, G or nothing; X₈ consists of F, L or I; X₉ consists of Sor T; X₁₀, X₁₁, X₁₂, and X₁₃ consist of S, I, L, G, V, A, or nothingcapable of reducing said TREM-1-mediated cell activation; b)administering said inhibitor to said patient under conditions such thatsaid at least one symptom is reduced. In one embodiment, the medicalcondition comprises sepsis. In one embodiment, the medical conditioncomprises NSCLC. In one embodiment, the medical condition comprisesinflammatory bowel disease. In one embodiment, the medical conditioncomprises acute mesenteric ischemia. In one embodiment, the medicalcondition comprises hemorrhagic shock. In one embodiment, the medicalcondition comprises a rheumatic disease. In one embodiment, therheumatic disease is selected from the group consisting of arthritis,ankylosing spondylitis, fibromyalgia, lupus, scleroderma, polymyositis,dermatomyositis, polymyalgia rheumatica, bursitis, tendinitis,vasculitis, carpal tunnel syndrome, complex regional pain syndrome,juvenile arthritis, Lyme disease, systemic lupus erythematosus, Kawasakidisease, fibromyalgia, and chronic fatigue syndrome.

In one embodiment, the present invention contemplates a drug deliverysystem comprising a variant TREM-1 transmembrane peptide conjugated to atherapeutic drug. In one embodiment, the variant peptide comprisessubstituted amino acids that optimize hydrophobicity relative to theTREM-1 transmembrane core peptide (Peptide 2, Table 2). In oneembodiment, the variant peptide comprises additional amino acids thatoptimize hydrophobicity relative to the TREM-1 transmembrane corepeptide (Peptide 2, Table 2). In one embodiment, the variant peptidecomprises additional and substituted amino acids that optimizehydrophobicity relative to the TREM-1 transmembrane core peptide(Peptide 2, Table 2). In one embodiment, the variant peptide comprisessubstituted amino acids that optimize helicity relative to the TREM-1transmembrane core peptide (Peptide 2, Table 2). In one embodiment, thevariant peptide comprises additional amino acids that optimize helicityrelative to the TREM-1 transmembrane core peptide (Peptide 2, Table 2).In one embodiment, the variant peptide comprises additional andsubstituted amino acids that optimize helicity relative to the TREM-1transmembrane core peptide (Peptide 2, Table 2). In one embodiment, thehelicity comprises inherent helicity. In one embodiment, the helicitycomprises induced helicity. In one embodiment, the helicity comprisesα-helices. In one embodiment, the therapeutic drug is selected from thegroup including, but not limited to, anti-cancer drugs,anti-inflammatory drugs, psychotropic drugs, anti-depressant drugs,stimulant drugs, anti-diabetic drugs, cardiovascular drugs,anti-thrombotic drugs, anti-proliferative drugs, or cytotoxic drugs.

In one embodiment, the present invention contemplates aprotease-resistance immunotherapeutic peptide comprising a variantTREM-1 transmembrane peptide. In one embodiment, the variant peptidecomprises at least one D-amino acid.

In one embodiment, the present invention contemplates a cyclicimmunotherapeutic peptide comprising a variant TREM-1 transmembranepeptide.

In one embodiment, the present invention contemplates a disulfide-linkeddimer of an immunotherapeutic peptide comprising a variant TREM-1transmembrane peptide.

In one embodiment, the present invention contemplates a medical devicecomprising a coating, wherein said coating comprises the peptidederivative of claim 1. In one embodiment, the coating further comprisesa polymer. In one embodiment, the polymer is selected from the groupincluding, but not limited to, phosphorylcholine, polyvinyl pyrrolidone,poly(acrylic acid), poly(vinyl acetamide), poly(propylene glycol),poly(ethylene co-vinyl acetate), poly(n-butyl methacrylate) orpoly(styrene-b-isobutylene-b-styrene). In one embodiment, the medicaldevice is selected from the group including, but not limited to, stents,grafts, catheters, endoscopes (i.e., for example, laparoscopes),atrial/venous fistulas, or cannulae.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates one embodiment of a MIRR-mediated transmembranesignal transduction utilizing the signaling chain homooligomerization(SCHOOL) model. The model indicates that the homooligomerization ofsignaling subunits has a role in triggering MIRR-mediated transmembranesignal transduction. Small unbroken black arrows indicate specificintersubunit hetero- and ho mo interactions between transmembrane andcytoplasmic domains. Circular arrow indicates ligand-induced receptorreorientation. Although it is not necessary to understand the mechanismof an invention, it is believed that ligand-induced MIRR clusteringleads to receptor reorientation and formation of a dimeric or oligomericintermediate in which signaling chains from different receptor unitsstart to trans-homointeract and form signaling oligomers. All interchaininteractions in this intermediate are denoted by broken black arrows,reflecting their transition state. It is further believed that upon theformation of signaling oligomers, protein tyrosine kinases phosphorylatethe tyrosine residues in the ITAMs (gray rectangles), leading totransmembrane transduction of activation signal, dissociation ofsignaling oligomers and internalization of the engaged MIRR-bindingdomains. Next, the signaling oligomers are believed to sequentiallyhomointeract with the relevant signaling subunits of non-engagedreceptors, resulting in the formation of higher signaling oligomers,thus propagating and amplifying the signals (not shown). This may leadto the release and subsequent internalization of the non-engagedligand-binding domains. A similar general scheme can be considered forthe pathway induced by receptor crosslinking, using antibodies tosignaling subunits (e.g. anti-CD3e or anti-Igb antibodies for TCRs andBCRs, respectively). Abbreviation: P, phosphate.

FIG. 2 illustrates one embodiment of a specific blockade oftransmembrane interactions between recognition and signaling subunitsresulting in “pre-dissociation” of the receptor complex, thus preventingformation of signaling oligomers and inhibiting antigen-dependent immunecell activation.

FIG. 3 illustrates one embodiment of a SCHOOL model of TREM-1-mediatedtransmembrane signal transduction and its inhibition. Solid arrows:Specific subunit homointeractions between cytoplasmic domains.

FIG. 4 illustrates one embodiment of a specific blockade oftransmembrane interactions between TREM-1 and DAP-12 resulting in“pre-dissociation” of a receptor complex.

FIG. 5 illustrates one embodiment of normal interactions between TREM-1and a DAP-12 subunit dimer to form a functional TREM-1/DAP-12 receptorcomplex.

FIG. 6 illustrates one embodiment of disrupted interactions betweenTREM-1 and DAP-12 resulting in a non-functional TREM-1/DAP-12 receptorcomplex.

FIG. 7 illustrates one embodiment of modulation of binding of the TREM-1Core and/or Extended peptides to the transmembrane domain of the DAP-12subunit dimer.

FIG. 8 presents various embodiments of TREM-1 peptide inhibitorsequences based upon a general formula, wherein in the general formuladescribes variants of the parent TREM-1 transmembrane sequence.

FIG. 9 shows one embodiment of a fluorescence polarization assay thatcould be used in high-throughput screening strategies to identify bothpeptide and nonpeptide inhibitors of protein-protein interaction betweenTREM-1 and DAP-12 subunits of the TREM-1/DAP-12 signaling complex. Grayoval: fluorophore; Arrow: transition vector of fluorescence emission.

FIG. 10 shows one embodiment of a fluorescence resonance energy transfer(FRET) assay that could be used in high-throughput screening strategiesto identify both peptide and non-peptide inhibitors of proteininteractions between TREM-1 and DAP-12 subunits of the TREM-1/DAP-12signaling complex. CFP: cyan fluorescent protein; YFP: yellowfluorescent protein.

FIG. 11 shows one embodiment of an enzyme-linked immunosorbent assay(ELISA) that could be used in high-throughput screening strategies toidentify both peptide and nonpeptide inhibitors of protein-proteininteraction between TREM-1 and DAP-12 subunits of the TREM-1/DAP-12signaling complex. HRP: horseradish peroxidase.

DEFINITIONS

The term “myeloid cell-mediated pathology” (or “myeloid cell-relatedpathologies”, or “myeloid cell-mediated disorder, or “myeloidcell-related disease”), as used herein, refers to any condition in whichan inappropriate myeloid cell response is a component of the pathology.The term is intended to include both diseases directly mediated bymyeloid cells, and also diseases in which an inappropriate myeloid cellresponse contributes to the production of abnormal antibodies,antibodies, as well as graft rejection.

The term “ligand-induced myeloid cell activation”, as used herein,refers to myeloid cell activation in response to the stimulation by thespecific ligand.

The term “stimulation”, as used herein, refers to a primary responseinduced by ligation of a cell surface moiety. For example, in thecontext of receptors, such stimulation entails the ligation of areceptor and a subsequent signal transduction event. With respect tostimulation of a myeloid cell, such stimulation refers to the ligationof a myeloid cell surface moiety that in one embodiment subsequentlyinduces a signal transduction event, such as binding the TREM-1/DAP-12complex. Further, the stimulation event may activate a cell andup-regulate or down-regulate expression or secretion of a molecule.

The term “ligand”, or “antigen”, as used herein, refers to a stimulatingmolecule that binds to a defined population of cells. The ligand maybind any cell surface moiety, such as a receptor, an antigenicdeterminant, or other binding site present on the target cellpopulation. The ligand may be a protein, peptide, antibody and antibodyfragments thereof, fusion proteins, synthetic molecule, an organicmolecule (e.g., a small molecule), or the like. Within the specificationand in the context of myeloid cell stimulation, the ligand (or antigen)binds the TREM receptor and this binding activates the myeloid cell.

The term “TREM receptor”, as used herein, refers to a member of TREMreceptor family: TREM-1, TREM-2, TREM-3 and TREM-4.

The term “activation”, as used herein, refers to the state of a cellfollowing sufficient cell surface moiety ligation to induce a noticeablebiochemical or morphological change. Within the context of myeloidcells, such activation, refers to the state of a myeloid cell that hasbeen sufficiently stimulated to induce production of interleukin 8(IL-8) and tumor necrosis factor alpha (TNF-alpha), differentiation ofprimary monocytes into immature dendritic cells, and enhancement ofinflammatory responses to microbial products. Within the context ofother cells, this term infers either up or down regulation of aparticular physico-chemical process.

The term “inhibiting myeloid cell activation” (or “TREM-mediated cellactivation”), as used herein, refers to the slowing of myeloid cellactivation, as well as completely eliminating and/or preventing myeloidcell activation.

The term, “treating a disease or condition”, as used herein, refers tomodulating myeloid cell activation including, but not limited to,decreasing cytokine production and differentiation of primary monocytesinto immature dendritic cells and/or slowing myeloid cell activation, aswell as completely eliminating and/or preventing myeloid cellactivation. Myeloid cell-related diseases and/or conditions treatable bymodulating myeloid cell activation include, but are not limited to,sepsis, non-small cell lung cancer, inflammatory bowel disease, acutemesenteric ischemia, hemorrhagic shock, rheumatic diseases such, forexample, as arthritis, ankylo sing spondylitis, fibromyalgia, lupus,scleroderma, polymyositis, dermatomyositis, polymyalgia rheumatica,bursitis, tendinitis, vasculitis, carpal tunnel syndrome, complexregional pain syndrome, juvenile arthritis, Lyme disease, systemic lupuserythematosus, Kawasaki disease, fibromyalgia, chronic fatigue syndrome,and other myeloid cell-related inflammatory conditions e.g. myositis,tissue/organ rejection.

The term, “subject” or “patient”, as used herein, refers to anyindividual organism. For example, the organism may be a mammal such as aprimate (i.e., for example, a human). Further, the organism may be adomesticated animal (i.e., for example, cats, dogs, etc.), livestock(i.e., for example, cattle, horses, pigs, sheep, goats, etc.), or alaboratory animal (i.e., for example, mouse, rabbit, rat, guinea pig,etc.).

The term, “therapeutically effective amount”, “therapeutically effectivedose” or “effective amount”, as used herein, refers to an amount neededto achieve a desired clinical result or results (inhibitingTREM-mediated cell activation) based upon trained medical observationand/or quantitative test results. The potency of any administeredpeptide or compound determines the “effective amount” which can vary forthe various compounds that inhibit myeloid cell activation (i.e., forexample, compounds inhibiting TREM ligand-induced myeloid cellactivation). Additionally, the “effective amount” of a compound may varydepending on the desired result, for example, the level of myeloid cellactivation inhibition desired. The “therapeutically effective amount”necessary for inhibiting differentiation of primary monocytes intoimmature dendritic cells may differ from the “therapeutically effectiveamount” necessary for preventing or inhibiting cytokine production.

The term, “agent”, as used herein, refers to any natural or syntheticcompound (i.e., for example, a peptide, a peptide variant, or a smallmolecule).

The term, “composition”, as used herein, refers to any mixture ofsubstances comprising a peptide and/or compound contemplated by thepresent invention. Such a composition may include the substancesindividually or in any combination.

The term, “intrinsic helicity”, as used herein, refers to the helicitywhich is adopted by a peptide in an aqueous solution.

The term, “induced helicity”, as used herein, refers to the helicitywhich is adopted by a peptide when in the presence of a helicityinducer, including, but not limited to, trifluoroethanol (TFE),detergents (e.g., sodium dodecyl sulfate, SDS) or lipids (e.g., lipidvesicles: small and large unilamellar vesicles, SUVs and LUVs,respectively, as described herein).

The term “therapeutic drug”, as used herein, refers to anypharmacologically active substance capable of being administered whichachieves a desired effect. Drugs or compounds can be synthetic ornaturally occurring, non-peptide, proteins or peptides, oligonucleotidesor nucleotides, polysaccharides or sugars. Drugs or compounds may haveany of a variety of activities, which may be stimulatory or inhibitory,such as antibiotic activity, antiviral activity, antifungal activity,steroidal activity, cytotoxic, cytostatic, anti-proliferative,anti-inflammatory, analgesic or anesthetic activity, or can be useful ascontrast or other diagnostic agents.

The term “effective dose” as used herein refers to the concentration ofany compound or drug contemplated herein that results in a favorableclinical response. In solution, an effective dose may range betweenapproximately 1 ng/ml-100 mg/ml, preferably between 100 ng/ml and 10mg/ml, but more preferably between 500 ng/ml and 1 mg/ml.

The term “administered” or “administering” a drug or compound, as usedherein, refers to any method of providing a drug or compound to apatient such that the drug or compound has its intended effect on thepatient. For example, one method of administering is by an indirectmechanism using a medical device such as, but not limited to a catheter,syringe etc. A second exemplary method of administering is by a directmechanism such as, local tissue administration (i.e., for example,extravascular placement), oral ingestion, transdermal patch, topical,inhalation, suppository etc.

The term “anti-inflammatory drug” means any compound, composition, ordrug useful for preventing or treating inflammatory disease.

The term “medical device”, as used herein, refers broadly to anyapparatus used in relation to a medical procedure. Specifically, anyapparatus that contacts a patient during a medical procedure or therapyis contemplated herein as a medical device. Similarly, any apparatusthat administers a drug or compound to a patient during a medicalprocedure or therapy is contemplated herein as a medical device. “Directmedical implants” include, but are not limited to, urinary andintravascular catheters, dialysis catheters, wound drain tubes, skinsutures, vascular grafts and implantable meshes, intraocular devices,implantable drug delivery systems and heart valves, and the like. “Woundcare devices” include, but are not limited to, general wound dressings,non-adherent dressings, burn dressings, biological graft materials, tapeclosures and dressings, surgical drapes, sponges and absorbablehemostats. “Surgical devices” include, but are not limited to, surgicalinstruments, endoscope systems (i.e., catheters, vascular catheters,surgical tools such as scalpels, retractors, and the like) and temporarydrug delivery devices such as drug ports, injection needles etc. toadminister the medium. A medical device is “coated” when a mediumcomprising an anti-inflammatory drug (i.e., for example, a variantTREM-1 transmembrane inhibitory peptide) becomes attached to the surfaceof the medical device. This attachment may be permanent or temporary.When temporary, the attachment may result in a controlled release of avariant TREM-1 transmembrane inhibitory peptide.

The term “endoscope” refers to any medical device that is capable ofbeing inserted into a living body and used for tasks including, but notlimited to, observing surgical procedures, performing surgicalprocedures, or applying medium to a surgical site. An endoscope isillustrated by instruments including, but not limited to, anarthroscope, a laparoscope, hysteroscope, cytoscope, etc. It is notintended to limit the use of an endoscope to hollow organs. It isspecifically contemplated that endoscopes, such as an arthroscope or alaparoscope is inserted through the skin and courses to a closedsurgical site.

The term “vascular access site” is defined herein as referring to anypercutaneous insertion of a medical device into the vasculature. Forexample, a hemodialysis catheter placement comprises a vascular accesssite. Such sites may be temporary (i.e., placed for a matter of hours)or permanent (i.e., placed for days, months or years).

The term “vascular graft” as used herein, refers to any conduit orportion thereof intended as a prosthetic device for conveying blood and,therefore, having a blood contacting surface (i.e., “luminal”). Whileusually in a tubular form, the graft may also be a sheet of materialuseful for patching portions of the circumference of living bloodvessels (these materials are generally referred to as surgical wraps).Likewise, the term vascular graft includes intraluminal grafts for usewithin living blood vessels. The inventive grafts as such may also beused as a stent covering on the exterior, luminal or both surfaces of animplantable vascular stent.

The term “synthetic vascular graft” as used herein, refers to anyartificial tube or cannula designed for insertion into a blood vessel.Such grafts may be constructed from polytetrafluoroethylene (PTFE).

The term “syringe” or “catheter” as used herein, refers to any device orapparatus designed for liquid administration, as defined herein. Asyringe or catheter may comprise at least one storage vessel (i.e., forexample, a barrel) wherein a single medium resides prior toadministration. A syringe or catheter comprising two or more barrels,each containing a separate medium, may mix the media from each barrelprior to administration or the media of each barrel may be administeredseparately. One of skill in the art will recognize that any catheterdesigned to perform dialysis, as defined herein, may also administerliquids.

The term “dialysis/apheresis catheter” as used herein, refers to anymulti-lumen catheter (i.e., for example, a triple lumen catheter)capable of providing a simultaneous withdrawal and return of blood to apatient undergoing a blood treatment process. Apheresis (called alsopheresis) comprises a blood treatment process involving separation ofblood elements that can remove soluble drugs or cellular elements fromthe circulation (Deisseroth et al., in Cancer: Principles And PracticeOf Oncology, Devita, V. T. Jr. et al. Editors, Philadelphia: J. B.Lippincott Company 1989, p. 2045-59). For example, blood is withdrawnfrom a donor, some blood elements (i.e., for example, plasma,leukocytes, platelets, etc.) are separated and retained. The unretainedblood elements are then retransfused into the donor.

The term “dialysis catheter” as used herein, refers to any devicecapable of removing toxic substances (impurities or wastes) from thebody when the kidneys are unable to do so. A dialysis catheter maycomprise a single catheter having at least a dual lumen (i.e., one lumenwithdraws arterial blood and a second lumen returns the dialyzed bloodto the venous system) or involve placing two catheters—one that isplaced in an artery, and one in an adjacent vein. Dialysis catheters aremost frequently used for patients who have kidney failure, but may alsobe used to quickly remove drugs or poisons in acute situations.

The term “peritoneal dialysis catheter” as used herein, refers to anycontinuous flow catheters with at least two lumens, one of which is ashort lumen (used to infuse a dialysis solution into the peritoneum),and the other of which is a long coiled lumen having a plurality ofopenings, generally located on the inside of the coil. It is believedthat peritoneal solutes enter into the coiled lumen openings and arethereby removed from the peritoneum. One hypothesis suggests thatperitoneal dialysis works by using the peritoneal membrane inside theabdomen as the semipermeable membrane. Special solutions that facilitateremoval of toxins may be infused in, remain in the abdomen for a time,and then drained out.

The term “fixed split-tip dialysis catheter” as used herein, refers toany catheter having at least two distinct elongated end portions thatextend substantially parallel to the longitudinal axis of the catheterand are flexible to the lateral displacement of an infused fluid. It isbelieved that this flexibility prevents a permanent catheter tip splaythat is known to injure tissue. Usually a fixed-tip dialysis catheterprovides indwelling vascular access for patients undergoing long-termrenal dialysis care (i.e., for example, end-stage renal disease).

The term “femoral catheter” as used herein, refers to any catheter thatis inserted into the femoral vein. Femoral catheters are typicallyprovided for intermediate term blood access because the superior venacava is relatively close to the right atrium of the heart, the minimalrange of shape changes of these veins with natural movements of thepatient (to minimize the damage to the vessel intima), and because ofgood acceptance by the patients of the skin exit on the thoracic wall.Further, the femoral veins are easy to cannulate, so that catheters ofthis invention may be inserted into the femoral veins at the bed side.

The term “attached” as used herein, refers to any interaction between amedium (or carrier) and a therapeutic drug. Attachment may be reversibleor irreversible. Such attachment includes, but is not limited to,covalent bonding, and non-covalent bonding including, but not limitedto, ionic bonding, Van der Waals forces or friction, and the like. Adrug is attached to a medium (or carrier) if it is impregnated,incorporated, coated, in suspension with, in solution with, mixed with,etc. The term “covalent bonding” as used herein, refers to an attachmentbetween two compounds (i.e., for example, a medium and a drug) thatcomprising a sharing of electrons.

As used herein, the term “peptide” refers to linear or cyclic orbranched compounds containing amino acids, amino acid equivalents orother non-amino groups, while still retaining the desired functionalactivity of a peptide. Peptide equivalents can differ from conventionalpeptides by the replacement of one or more amino acids with relatedorganic acids such as p35 aminobenzoic acid (PABA), amino acid analogs,or the substitution or modification of side chains or functional groups.Peptide equivalents encompass peptide mimetics or peptidomimetics, whichare organic molecules that retain similar peptide chain pharmacophoregroups as are present in the corresponding peptide. The term “peptide”refers to peptide equivalents as well as peptides. The amino acids canbe in the L or D form so long as the binding function of the peptide ismaintained.

As used herein, the term “cyclic peptide” refers to a peptide having anintramolecular bond between two non-adjacent amino acids. Thecyclization can be effected through a covalent or non-covalent bond.Intramolecular bonds include, but are not limited to, backbone tobackbone, side-chain to backbone and side-chain to side-chain bonds.

As used herein, the term “dimer” as applied to peptides refers tomolecules having two peptide chains associated covalently ornon-covalently, with or without linkers. Peptide dimers wherein thepeptides are linked C-terminus to N-terminus may also be referred to as“tandem repeats” or “tandem dimers.” Peptide dimers wherein the peptidesare linked C- to C-terminus, or N- to N-terminus may also be referred toas “parallel repeats” or “parallel dimers.”

The term “placing” as used herein, refers to any physical relationship(i.e., secured or unsecured) between a patient's biological tissue and asurgical material, wherein the surgical material comprises apharmaceutical drug that may be, optionally, attached to a medium. Sucha physical relationship may be secured by methods such as, but notlimited to, gluing, suturing, stapling, spraying, laying, impregnating,and the like. The term “parts by weight”, as used herein, when used inreference to a particular component in a composition denotes the weightrelationship between the component and any other components in thecomposition for which a pan by weight is expressed.

The term “protecting groups”, as used herein, refer to those groupswhich prevent undesirable reactions (such as proteolysis) involvingunprotected functional groups. In one embodiment, the present inventioncontemplates that the protecting group is an acyl or an amide. In oneembodiment, the acyl is acetate. In another embodiment, the protectinggroup is a benzyl group. In another embodiment, the protecting group isa benzoyl group. The present invention also contemplates combinations ofsuch protecting groups.

The term “protein”, as used herein, refers to compounds comprising aminoacids joined via peptide bonds and includes proteins and polypeptides;and may be an intact molecule, a fragment thereof, or multimers oraggregates of intact molecules and/or fragments; and may occur in natureor be produced, e.g., by synthesis (including chemical and/or enzymatic)or genetic engineering. The terms “protein” and “polypeptide” are usedherein interchangeably.

As used herein, where “amino acid sequence” is recited herein to referto an amino acid sequence of a protein molecule. An “amino acidsequence” can be deduced from the nucleic acid sequence encoding theprotein. However, terms such as “polypeptide” or “protein” are not meantto limit the amino acid sequence to the deduced amino acid sequence, butinclude posttranslational modifications of the deduced amino acidsequences, such as amino acid deletions, additions, and modificationssuch as glycosylation and addition of lipid moieties.

The term “portion” when used in reference to a protein (as in “a portionof a given protein”) refers to fragments of that protein. The fragmentsmay range in size from four amino acid residues to the entire aminosequence minus one amino acid.

The term “analog”, as used herein, includes any peptide having an aminoacid sequence substantially identical to one of the sequencesspecifically shown herein in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the abilities as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another,the substitution of one polar (hydrophilic) residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

The term “conservative substitution”, as used herein, also includes theuse of a chemically derivatized residue in place of a non-derivatizedresidue provided that such peptide displays the requisite inhibitoryfunction on myeloid cells as specified herein. The term derivativeincludes any chemical derivative of the peptide of the invention havingone or more residues chemically derivatized by reaction of side chainsor functional groups.

The term “homolog” or “homologous” when used in reference to apolypeptide refers to a high degree of sequence identity between twopolypeptides, or to a high degree of similarity between thethree-dimensional structure or to a high degree of similarity betweenthe active site and the mechanism of action. In a preferred embodiment,a homolog has a greater than 60% sequence identity, and more preferablygreater than 75% sequence identity, and still more preferably greaterthan 90% sequence identity, with a reference sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as, for example, bythe programs SIM+LALNVIEW, LALIGN and DIALIGN (expasy.ch/tools) usingdefault gap weights, share at least 80 percent sequence identity,preferably at least 90 percent sequence identity, more preferably atleast 95 percent sequence identity or more (e.g., 99 percent sequenceidentity). Preferably, residue positions which are not identical differby conservative amino acid substitutions.

The terms “variant” and “mutant” when used in reference to a polypeptiderefer to an amino acid sequence that differs by one or more amino acidsfrom another, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One type of conservative amino acidsubstitutions refers to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions (inother words, additions), or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Preferred variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

It is understood by the person of ordinary skill in the art that theterms “TREM1_HUMAN”, “TREM-1 receptor”, “TREM-1 receptor subunit”,“TREM-1 subunit”, and “TREM-1 recognition subunit” refer to thenaturally occurring human protein listed in the UniProt Knowledgebase(UniProtKB, uniprot.org) under the name “TREM1_HUMAN”. The protein aminoacid sequence can be found under the entry UniProt KB/Swiss-Prot Q9NP99.It is further understood that the terms “TREM1_Mouse”, “mouse TREM-1receptor”, “mouse TREM-1 receptor subunit”, “mouse TREM-1 subunit”, and“mouse TREM-1 recognition subunit” refer to the naturally occurringmouse protein listed in the UniProt Knowledgebase (UniProtKB,uniprot.org) under the name “TREM1_MOUSE”. The protein amino acidsequence can be found under the entry UniProt KB/Swiss-Prot Q9JKE2. Itis still further understood that the terms “TREM1_Bovin”, “bovine TREM-1receptor”, “bovine TREM-1 receptor subunit”, “bovine TREM-1 subunit”,and “bovine TREM-1 recognition subunit” refer to the naturally occurringbovine protein listed in the UniProt Knowledgebase (UniProtKB,uniprot.org) under the name “TREM1_BOVIN”. The protein amino acidsequence can be found under the entry UniProt KB/Swiss-Prot Q6QUN5. Itis further understood that the terms “TREM1_PIG”, “pig TREM-1 receptor”,“pig TREM-1 receptor subunit”, “pig TREM-1 subunit”, and “pig TREM-1recognition subunit” refer to the naturally occurring pig protein listedin the UniProt Knowledgebase (UniProtKB, uniprot.org) under the name“TREM1_PIG”. The protein amino acid sequence can be found under theentry UniProt KB/Swiss-Prot Q6TY16.

It is understood by the person of ordinary skill in the art that theterms “TYOBP_HUMAN”, “DAP-12”, “DAP-12 subunit”, and “DAP-12 signalingsubunit” refer to the naturally occurring human protein listed in theUniProt Knowledgebase (UniProtKB, uniprot.org) under the name“TYOBP_HUMAN”. The protein amino acid sequence can be found under theentry UniProt KB/Swiss-Prot 043914. It is further understood that theterms “TYOBP_MOUSE”, “mouse DAP-12”, “mouse DAP-12 subunit”, and “mouseDAP-12 signaling subunit” refer to the naturally occurring mouse proteinlisted in the UniProt Knowledgebase (UniProtKB, uniprot.org) under thename “TYOBP_MOUSE”. The protein amino acid sequence can be found underthe entry UniProt KB/Swiss-Prot 054885. It is still further understoodthat the terms “TYOBP_BOVIN”, “bovine DAP-12”, “bovine DAP-12 subunit”,and “bovine DAP-12 signaling subunit” refer to the naturally occurringbovine protein listed in the UniProt Knowledgebase (UniProtKB,uniprot.org) under the name “TYOBP_BOVIN”. The protein amino acidsequence can be found under the entry UniProt KB/Swiss-Prot Q95J79. Itis further understood that the terms “TYOBP_RAT”, “rat DAP-12”, “ratDAP-12 subunit”, and “rat DAP-12 signaling subunit” refer to thenaturally occurring rat protein listed in the UniProt Knowledgebase(UniProtKB, uniprot.org) under the name “TYOBP_RAT”. The protein aminoacid sequence can be found under the entry UniProt KB/Swiss-Prot Q6X9T7.It is further understood that the terms “TYOBP_PIG”, “pig DAP-12”, “pigDAP-12 subunit”, and “pig DAP-12 signaling subunit” refer to thenaturally occurring pig protein listed in the UniProt Knowledgebase(UniProtKB, uniprot.org) under the name “TYOBP_PIG”. The protein aminoacid sequence can be found under the entry UniProt KB/Swiss-Prot Q9TU45.It is further understood that the terms “Q95KS5_SHEEP”, “sheep DAP-12”,“sheep DAP-12 subunit”, and “sheep DAP-12 signaling subunit” refer tothe naturally occurring sheep protein listed in the UniProtKnowledgebase (UniProtKB, uniprot.org) under the name “Q95KS5_SHEEP”.The protein amino acid sequence can be found under the entry UniProtKB/Swiss-Prot Q95KS5.

DETAILED DESCRIPTION OF THE INVENTION

The peptides and compositions of the present invention are derived fromamino acid sequence of transmembrane regions of TREM-1 receptor, can bedesigned and formulated to be delivered orally, optimized for theirefficacy and specificity in accordance to the suggested criteria, thusimproving upon prior art and overcoming current limitations in the priorart.

Additional advantages of the invention will be set forth in part in thedescription that follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

The present invention relates to peptides and compounds which affect theactivating TREM receptor signaling pathway in granulocytes, monocytes,macrophages, neutrophils, microglia, dendritic cells, osteoclasts,platelets and other cells. The present invention further relates to thetreatment or prevention of cancer, allergic diseases, inflammatory boweldisease, acute mesenteric ischemia, hemorrhagic shock, autoimmunediseases, including but not limited to, rheumatoid arthritis and otherrheumatic diseases, sepsis and other inflammatory or other conditioninvolving myeloid cell activation, and, more particularly, TREMreceptor-mediated cell activation. Specifically, the peptides andcompounds are useful in the treatment and/or prevention of a disease orcondition where myeloid cells are involved or recruited. The peptides ofthe present invention also are useful in the production of medicaldevices comprising peptide matrices (for example, medical implants andimplantable devices). In one embodiment, TREM-1/DAP-12 receptor complexsignaling is inhibited by variant peptides binding to the transmembraneregion of the DAP-12 subunit.

Various methods of application are proposed to use these proteinvariants including, but not limited to; i) treating diseases or othermedical conditions where myeloid cells are involved or recruited; ii)drug delivery systems; iii) a sequence-based rational drug designmethod; iv) protease-resistance immunotherapeutic peptides; v) coatingsof medical devices, such as implants and implantable devices.

The present invention contemplates constructing a series of variantpeptides homologous to a transmembrane core sequence of the triggeringreceptor expressed on myeloid cells-1 (TREM-1) receptor and capable ofreducing said myeloid cell activation by action on the activating TREM-1receptor. The TREM-1/DAP-12 receptor signaling complex is composed ofTREM-1 recognition subunit and a DAP-12 signaling subunit dimer. Thus,TREM-1 is a member of family of multichain immune recognition receptors(MIRRs) which are characterized by a common and distinct receptorarchitectural feature—their ligand-binding subunits and signalingsubunits represent separate transmembrane protein chains that arenoncovalently bound in the transmembrane milieu (Sigalov A. B. TrendsImmunol 2004; 25:583-9; Sigalov A. B. Adv Exp Med Biol 2008;640:268-311; Sigalov A. B. Adv Exp Med Biol 2008; 640:121-63; Keegan A.D. & Paul W. E. Immunol Today 1992; 13:63-8). The DAP-12 signalingsubunit has a conserved single negative charge in its transmembrane (TM)domain (TABLE 1), while TM domain of the TREM-1 recognition subunitcontains one positive charge (TABLE 2). The integrity and functionallyof the receptor is provided by the TM electrostatic interactions. Thepositively charged Lys residue in the TM region of the TREM-1 chaininteracts with the negatively charged Asp residues of the TM domains ofDAP-12 homodimer. Assembly of other TREM receptors is similar. Recently,these interactions have been suggested as universal therapeutic targetsfor a diverse variety of pathologies (Sigalov A. B. Trends Immunol 2004;25:583-9; Sigalov A. B. Adv Exp Med Biol 2007; 601:335-44).

The TREM-1/DAP-12-coupled receptor signaling pathway resident withinmyeloid cell membranes represents but one mechanism responsible formyeloid cell activation. Although it is not necessary to understand themechanism of an invention, it is believed that these variant peptidesinsert themselves into the cell membrane and act as a “receptor decoy”for TREM ligand molecules. It is further believed thatTREM-1/DAP-12-mediated cell activation requires the bridging of multipleTREM-1/DAP-12-coupled receptors that generates an intracellularactivation signal by bringing membrane-embedded DAP-12 subunits intoclose proximity and correct (permissive) orientation. These TREM-1-likepeptide variants may prevent cell activation by reducing DAP-12-DAP-12aggregation by generating TREM-1/DAP-12/peptide variant bridges in thepresence of ligand. It is further believed that the molecular basis forthe prevention of myeloid cell activation is based upon protein-proteininteractions.

Protein-protein interactions are involved in most biological processesand thus represent an appealing target for innovative drug development.These interactions can be targeted by small molecule inhibitors,peptides, and peptidomimetics. Consequently, indirect protein therapythat alters protein-protein interactions represents an alternative todirect protein therapeutics (i.e., for example, immunotherapy) andavoids dangerous side effects. Indirectly acting peptides may serve asactive regulators and participate in molecular cross talk, which drivesmetabolic processes. These indirectly acting peptides are also extremelypotent, showing high specificity, and have few toxicological problems.Moreover, these indirectly acting peptides do not accumulate in organsor suffer from drug-drug interactions as many small molecules do. Theycan be used as therapeutic agents, or as a starting point for developingpeptidomimetics and small molecular weight inhibitors.

1. Triggering Receptors Expressed on Myeloid Cells

The myeloid lineage gives rise to cells that function both in the immunesystem and in remodeling host tissue (Klesney-Tait et al. Nat Immunol2006; 7:1266-73). The differentiation and activation of those cells isregulated by signals received through the cell surface and intracellularreceptors that recognize both soluble and surface-expressed ligands.Those receptors can be broadly classified into two groups: those‘tasked’ to recognize a specific event, and those through which the cellreceives more general information about the state of the organism.Examples of the former include the Toll-like receptors (TLRs), whichsignal the presence of a microbial pathogen, and growth factor receptor,which drives differentiation. Those task-specific receptors determinethe qualitative nature of the response of the cells, such as activationor differentiation. In contrast, stimulation of receptors in the secondgroup (those that ‘read out’ the state of the organism) does not byitself induce a substantial response by the cell but instead helps toset thresholds for the cellular response to specific stimuli and tomodulate the magnitude of that response. By integrating the responses ofboth groups of receptors, the cell can generate a carefully gradedresponse to a specific set of conditions.

TREM proteins (‘triggering receptors expressed on myeloid cells’) are afamily of cell surface receptors expressed broadly on myeloid cells. Thefirst TREM identified (TREM-1) was characterized as an amplifier of theimmune response that strongly potentiates the activation of leukocytesin response to microbial products. The TREM family has since beenextended to include proteins expressed on granulocytes, monocytes,macrophages, microglia, dendritic cells, osteoclasts and platelets.Generally, these receptors seem to function mainly as modulators of thecellular response, falling squarely into the second group of receptors(those that set ‘thresholds’ for cells) described above. By coordinatingdiverse stimuli, TREM proteins have both positive and negative functionsin regulating the activation and differentiation of myeloid cells.

The TREM family includes at least two activating receptors, namelyTREM-1 and TREM-2 which are transmembrane glycoproteins with a singleextracellular Ig-like domain, a transmembrane region with a charged Lysresidue, and a short intracellular region (Gibot S. Crit Care 2005;9:485-9). Engagement of TREMs, after association with the signalingadapter protein DAP12 (which contains an immunoreceptor tyrosine-basedactivation motif, ITAM), triggers a signaling pathway involvingζ-chain-associated protein 70 (ZAP70) and spleen tyrosine kinase. Thisin turn leads to the recruitment and tyrosine phosphorylation of adaptormolecules such as growth factor receptor binding protein 2, andactivation of phosphatidylinositol 3-kinase, phospholipase C-gamma,extracellular signal regulated kinase-1 and -2, and p38mitogen-associated protein kinase. Activation of these pathways leads tointracellular calcium mobilization, actin cyto skeleton rearrangement,and activation of transcription factors. TREM-1 has been implicated inmounting the inflammatory response, whereas TREM-2 regulates dendriticcells, osteoclasts and microglia.

TREM receptors are attractive targets for therapy of myeloidcell-related pathologies. TREM-1 seems particularly attractive in thisrespect. Since its detection, the function of TREM-1 and the signaltransduction pathway induced by the TREM-1/DAP-12 receptor signalingcomplex have been extensively studied. The selective inhibition ofTREM-1 and/or its signaling is thought by most workers in the field toprovide new promising therapeutic strategies to fight myeloidcell-mediated disease (Bouchon et al. J Immunol 2000; 164:4991-5;Bouchon et al. Nature 2001; 410:1103-7; Gibot S. Crit Care 2005;9:485-9; Gibot et al. J Exp Med 2004; 200:1419-26; Gibot et al. Shock2009; 32:633-7; Ling et al. Chinese Med J 2010; 123:1561-5; Gibot et al.Crit Care Med 2008; 36:504-10; Klesney-Tait et al. Nat Immunol 2006;7:1266-73; Murakami et al. Arthritis Rheum 2009; 60:1615-23; Sharif 0. &Knapp S. Immunobiology 2008; 213:701-13). In addition, recent findingshave linked TREM-1 to non-small cell lung cancer (NSCLC) (Ho et al. Am JRespir Crit Care Med 2008; 177:763-70) and inflammatory bowel disease(IBD) (Ford J. W. & McVicar D. W. Curr Opin Immunol 2009; 21, 38-46).

Currently, very few approaches have pursued an inhibition ofTREM-1-mediated transmembrane signaling. Antibodies that specificallyrecognize TREM-1 were used to prevent ligand binding and initiation ofcell aggregation (US Pat Appl 20080247955; Piccio et al. Eur J Immunol2007; 37, 1290-301; herein incorporated by reference in their entirety).Fusion proteins between human IgG1 constant region and the extracellulardomain of mouse TREM-1 or that of human TREM-1 were also suggested toprevent ligand binding (US Pat Appls 20090081199 and 20030165875).However, these large protein molecules pose serious disadvantages.

Peptides based on TREM-1-derived sequences for disrupting TREM-1function presumably by blocking binding of the receptor with its cognateligand have also been disclosed (US Pat Appl 20060246082) or published(Murakami et al. Arthritis Rheum 2009; 60:1615-23; Gibot et al. CritCare Med 2008; 36:504-10; Gibot et al. Shock 2009; 32:633-7). Despitemultiple advantages of these peptides as compared to antibodies, theyhave relatively low efficacy in terms of inhibiting TREM-1, thus havinga high potential for toxicity and side effects. For example, thesystemic administration of a synthetic antagonistic TREM-1 peptide thatmimics short highly interspecies-conserved extracellular domains ofTREM-1 and is often called as LP-17 suppresses collagen-inducedarthritis, although the effect is not as complete as that observedfollowing viral gene transfer (Murakami et al. Arthritis Rheum 2009;60:1615-23).

2. SCHOOL Model of TREM Signaling

Further development of effective therapeutic agents which preventTREM-mediated cell activation depends on an improved understanding ofthe TREM-1/DAP-12-coupled receptor signaling pathway. Upon stimulationby ligand, TREM-1 signals through a noncovalently associated DAP-12, atransmembrane protein that mediates signaling through its ITAM.

In this regard, a TREM-1 receptor is a member of the MIRR family,members of which are multisubunit complexes formed by the association ofrecognition subunits with ITAM-containing signaling subunits. Thisassociation in resting cells is mostly driven by the noncovalenttransmembrane interactions between recognition and signaling componentsand plays a role in receptor assembly and integrity. Ligand bindingresults in phosphorylation of the ITAM tyrosines, which triggers theelaborate intracellular signaling cascade. The mechanism linkingextracellular clustering of MIRR ligand-binding subunits tointracellular phosphorylation of signaling subunits remains to beidentified. In this regard, the mechanisms of TREM-1 transmembranesignaling has been also elusive, thus hindering the further developmentof promising therapeutic strategies for the treatment/prophylaxis ofmyeloid cell-mediated disease.

Therapeutic strategies contemplated herein involve MIRR triggering andsubsequent signaling. MIRR-mediated signal transduction, their role inhealth and disease, and the use of these receptors as attractive targetsfor rational drug design efforts in the treatment of several immunedisorders are described in (US Pat Appl 20090075899; Sigalov A. SeminImmunol 2005; 17:51-64; Sigalov A. B. Trends Immunol 2004; 25:583-9;Sigalov A. B. Trends Pharmacol Sci 2006; 27:518-24; Sigalov A. B. AdvExp Med Biol 2007; 601:335-44; Sigalov A. B. Adv Exp Med Biol 2008;640:268-311) which are incorporated herein by reference in theirentirety.

In one embodiment, the present invention contemplates therapeutictargets compatible with a novel model of MIRR signaling; the SignalingChain HomO-OLigomerization (SCHOOL) model (See FIG. 1) (Sigalov A. B.Trends Immunol 2004; 25:583-9; Sigalov A. B. Trends Pharmacol Sci 2006;27:518-24; Sigalov A. B. Adv Exp Med Biol 2008; 640:268-311; Sigalov A.B. Adv Exp Med Biol 2008; 640:121-63; incorporated herein by referencein their entirety). Although it is not necessary to understand themechanism of an invention, it is believed that the structural similarityof the MIRRs provides the basis for the similarity in the mechanisms ofMIRR-mediated signaling (FIG. 1). It is also believed that the modelreveals MIRR transmembrane interactions as new therapeutic targets (SeeFIG. 2). It is further believed that a general pharmaceutical approachbased upon this SCHOOL model can be used to treat diverseimmune-mediated diseases.

Application of the SCHOOL model to the transmembrane signal transductionmediated by a TREM receptor (i.e., for example, TREM-1) (FIG. 3)suggested that an inhibition of TREM-1/DAP-12 signaling may be achievedby using transmembrane-targeted agents which specifically disrupttransmembrane interactions between TREM-1 and DAP-12 subunits (FIG. 4).For example, the simplest agents would be synthetic peptidescorresponding to the TREM-1 transmembrane domain. Without being limitedby a particular theory, the basic principles of one proposed mechanismby which peptides and other compound of the present invention may workby TREM-1-mediated transmembrane signaling. See, FIGS. 4, 5, and 6.

It is believed that ligand-induced clustering of a TREM-1/DAP-12receptor complex leads to formation of DAP-12 signaling oligomers withsubsequent phosphorylation of the ITAM Tyr residues and cell activation.See, FIG. 3. This hypothesis suggests that a TREM-1 Core Peptide(TREM-1-CP), a peptide corresponding to the transmembrane region ofTREM-1, inserts into the cell membrane and competitively binds to thetransmembrane domain of DAP-12 chain, thus displacing a TREM-1 receptorfrom interacting with a signaling DAP-12 subunit, thereby resulting in a“pre-dissociation” of a TREM-1/DAP-12 receptor complex. As aconsequence, ligand-induced TREM-1 clustering does not lead to formationof DAP-12 signaling oligomers and subsequent cell activation. See, FIG.4.

Normal transmembrane interactions between the TREM-1 and the DAP-12dimer forming a functional TREM-1/DAP-12 receptor complex comprisepositively charged lysine amino acid within the TREM-1 transmembraneportion and negatively charged aspartic acid pairs in a DAP-12 dimer,thereby allowing subunit association. See FIG. 5. Although it is notnecessary to understand the mechanism of an invention, it is believedthat interactions between a lysine residue of a TREM-1 core peptideinhibitor and an aspartic acid residue of a DAP-12 dimer disrupt thetransmembrane interactions between TREM-1 and DAP-12, thereby“disconnecting” TREM-1 and resulting in a non-functioning receptor. SeeFIG. 6.

3. TREM-1 Peptides and Variants Thereof

Although it is not necessary to understand the mechanism of aninvention, it is believed that a hydrophobic/polar/charged amino acidsequence patterning, rather than sequence similarity, within thetransmembrane TREM-1 domain plays a dominant role in the development ofeffective peptide-based inhibitors of TREM-1-mediated cell activation.For example, despite the lack of sequence similarity, the fusion peptide(FP) in the N terminus of the HIV envelope glycoprotein 41, inhibit Tcell receptor (TCR)-mediated T cell activation in vitro and in vivo in asimilar way but more effectively than the transmembrane TCR core peptide(CP) with 100-fold lower the 50% inhibitory concentration (IC50) valuesfor FP than those observed for CP.

In some embodiments, as contemplated by the present invention, optimalpeptide inhibitors and peptide inhibitor analogues are designed usinghydrophobic/polar/charged sequence pattern criteria and associatedevaluation techniques. These peptide inhibitors may then be synthesizedand tested in cell function inhibition assays and in animal studies.

Listed below in Table 1 are reported transmembrane sequences of TREM-1and DAP-12 in a number of species. These regions are highly conservedand the substitutions between species are very conservative. Thissuggests a functional role for the transmembrane regions of both, TREM-1and DAP-12, constituents of the complex. These regions strongly interactbetween themselves, thus maintaining the integrity of the TREM-1/DAP-12receptor signaling complex in resting cells. These transmembrane domainsare short and should be easily mimicked by synthetic peptides andcompounds. Based on these features, and taking advantage of the SCHOOLmodel of MIRR signaling to explain TREM-1-mediated cell activation, thepresent invention contemplates a new approach of intervening andmodulating TREM-1 function. In some embodiments, synthetic peptides andcompounds are contemplated that may provide successful treatment optionsin the clinical setting.

TABLE 1  Sequence comparison of TREM-1 and DAP-12 transmembrane regions (accession codes are given in parenthesis) SEQUENCE SPECIES TREM-1DAP-12 HUMAN: SEQ ID NO: 4 SEQ ID NO: 5 (O43914)IVILLAGGFLSKSLVFSVLFA (Q9NP99) GVLAGIVMGDLVLTVLIALAV MOUSE: SEQ ID NO: 6SEQ ID NO: 7 (O54885) VTISVICGLLSKSLVFIILFI (Q9JKE2)GVLAGIVLGDLVLTLLIALAV BOVIN: SEQ ID NO: 8 SEQ ID NO: 9 (Q95J79)IIIPAACGLLSKTLVFIGLFA (Q6QUN5) GVLAGIVLGDLMLTLLIALAV SHEEP: not knownSEQ ID NO: 10 (Q95KS5) GVLAGIVLGDLMLTLLIALAV RAT: not knownSEQ ID NO: 11 (Q6X9T7) GVLAGIVLGDLVLTLLIALAV PIG: SEQ ID NO: 12SEQ ID NO: 13 (Q9TU45) ILPAVCGLLSKSLVFIVLFVV (Q6TYI6)GILAGIVLGDLVLTLLIALAV CLUSTAL W 2.0 multiple sequence alignment: HUMANIVILLAGGFLSKSLVFSVLFA— 21 GVLAGIVMGDLVLTVLIALAV 21 (SEQ ID NOS: 4, 5)MOUSE VTISVICGLLSKSLVFIILFI— 21GVLAGIVLGDLVLTLLIALAV 21 (SEQ ID NOS: 6, 7) BOVIN IIIPAACGLLSKTLVFIGLFA—21 GVLAGIVLGDLMLTLLIALAV 21 (SEQ ID NOS: 8, 9) SHEEP —GVLAGIVLGDLMLTLLIALAV 21 (SEQ ID NO: 10) RAT —GVLAGIVLGDLVLTLLIALAV 21 (SEQ ID NO: 11) PIG —ILPAVCGLLSKSLVFIVLFVV 21GILAGIVLGDLVLTLLIALAV 21 (SEQ ID NOS: 12, 13)   :    *:***:***  ***:*****:***:**:******

In one embodiment, the present invention contemplates a series ofpeptides that are inhibitors of a TREM receptor (i.e., for example, aTREM-1/DAP-12 complex) Although it is not necessary to understand themechanism of an invention, it is believed that this inhibition ismediated by disrupting the transmembrane interactions between therecognition, TREM-1, and signaling, DAP-12, subunits. In otherembodiments, these peptide inhibitors treat and/or prevent diseasesand/or conditions comprising activation of TREM-expressing myeloid cell.In one embodiment, the peptide inhibitors modulate TREM-1-mediated cellactivation. In another embodiment, the present invention contemplates adrug delivery system (e.g., as disclosed in US Pat Appl 20080193375 andincorporated herein by reference in its entirety) comprising peptideinhibitors of the present invention. As disclosed in PCT Appl. No.PCT/US2010/52117 “Methods and Compositions for Targeted Imaging” theentire content of which is incorporated herein by reference, in variousconfigurations, a drug delivery composition can also compriselipoprotein nanoparticles wherein said nanoparticles comprise at leastone modified apolipoprotein and at least one lipid. Although it is notnecessary to understand the mechanism of an invention, it is believedthat the peptide inhibitor drug delivery system functions by penetratingthe cell membrane.

Sequence-based rational design can be used as a tool in order toincrease the effectiveness of the peptides to inhibit the function ofthe TREM-1/DAP-12 receptor complex. For example, a conservative aminoacid substitution of lysine for arginine or insertion of at least onesupplemental positively charged amino acid residue (i.e., for example,arginine and/or lysine) may be made in certain locations on α-helixes ofTREM-1 core or extended peptides. Although it is not necessary tounderstand the mechanism of an invention, it is believed that thesechanges should result in increased binding activity to the transmembranedomain of the DAP-12 signaling subunit dimer, thus enhancing theeffectiveness of the peptides to inhibit the function of anTREM-1/DAP-12 receptor complex. See FIG. 7.

TREM-1 peptide inhibitors and variants thereof contemplated herein maybe modified at the carboxy terminal without loss of activity.Accordingly, it is intended that the present invention includes withinits scope, peptides which include additional amino acids to the “core”sequence of the peptide of the present invention and which affect theinteraction of TREM-1 and DAP-12 subunits of the TREM-1/DAP-12 complex(i.e., for example, an Extended Peptide).

In some embodiments, the peptide inhibitors comprise D-stereoisomericamino acids, thereby allowing the formulation of immunotherapeuticpeptides with increased resistance to protease degradation. In oneembodiment, the D-amino acid peptide inhibitors are used for theclinical treatment in myeloid cell-mediated disorders. Although it isnot necessary to understand the mechanism of an invention, it isbelieved that these peptide inhibitors prevent activation ofTREM-expressing myeloid cells.

In some embodiments, the present invention contemplate peptideinhibitors that are protease resistant. In one embodiment, suchprotease-resistant peptide inhibitors are peptides comprising protectinggroups. For example, a peptide may be protected from exoproteinasedegradation by N-terminal acetylation (“Ac”) and/or C-terminalamidation.

In some embodiments, the peptide inhibitors comprise conjugated lipidsand/or sugars. In other embodiments, the peptide inhibitors comprisehydrophobic amino acid motifs, wherein said motifs are known to increasethe membrane penetrating ability of peptides and proteins. Although itis not necessary to understand the mechanism of an invention, it isbelieved that either lipid/sugar conjugation and/or hydrophobic aminoacid motifs increase the efficacy of TREM-1 inhibition using eitherTREM-1 Core Peptides and/or Extended Peptides.

In some embodiment, the peptides and compounds contemplated by thepresent invention may be used for production ofpeptide/compound-containing implants or implantable devices.

4. TREM-1 Transmembrane Segments

A. Transmembrane Peptide Variants for Inhibition of TREM-1 Signaling

The present invention described herein relates to unknown syntheticpeptides and derivatives thereof, which may be useful in the clinicaltreatment and/or prevention of myeloid cell-mediated disorders.

In one embodiment, the present invention contemplates a peptidederivative having the general formula R₁-A-B-C-D-E-F-G-H-R₂ (See FIG. 8)(SEQ ID NO: 14) or a disulfide-bridged, linear dimer thereof, or acyclic dimer thereof, wherein:

A is absent, Ser, Thr, or a peptide consisting of 1 to 6 hydrophobicuncharged D- or L-amino acids, or a peptide consisting of 1 to 6hydrophobic uncharged D- or L-amino acids surrounding a positivelycharged D- or L-amino acid which is spaced 6 amino acids from E;

B is a peptide consisting of 1 to 2 non-hydrophobic uncharged D- orL-amino acids, including D- or L-cysteine or a D- or L-cysteinehomologue, or a peptide consisting of 1 to 2 non-hydrophobic unchargedD- or L-amino acids, including D- or L-cysteine or a D- or L-cysteinehomologue surrounding a positively charged D- or L-amino acid which isspaced 3 amino acids from E;

C is a peptide consisting of 1 to 2 hydrophobic uncharged D- or L-aminoacids;

D is a non-hydrophobic uncharged D- or L-amino acid, including D- orL-cysteine or a D- or L-cysteine homologue;

E is a positively charged D- or L-amino acid;

F is a non-hydrophobic uncharged D- or L-amino acid, including D- orL-cysteine or a D- or L-cysteine homologue;

G is a peptide consisting of 1 to 3 hydrophobic uncharged D- or L-aminoacids, or a peptide consisting of 1 to 3 hydrophobic uncharged D- orL-amino acids surrounding a positively charged D- or L-amino acid whichis spaced 3 amino acids from E;

H is absent, Ser, Thr, or a peptide consisting of 1 to 6 hydrophobicuncharged D- or L-amino acids, or a peptide consisting of 1 to 6hydrophobic uncharged D- or L-amino acids surrounding a positivelycharged D- or L-amino acid which is spaced 6 amino acids from E;

R₁ is absent (i.e., for example, -H) or 1-amino-glucose succinate,2-aminododecanoate, or myristoylate; and

R₂ is absent (i.e, for example, -H) or Gly-Tris-monopalmitate,-dipalmitate and -tripalmitate.

In some embodiments, peptide derivatives are created wherein (SEQ ID NO:105):

-   -   A is selected from the group comprising Ser, Leu, Val, Ala, Arg,        and Lys; B is selected from the group comprising Gln, Ser, Gly,        Cys, and Arg;    -   C is selected from the group comprising Pro, Phe, Ala and Leu;    -   D is selected from the group comprising Ser, Gly, and Thr;    -   E is selected from Arg or Lys;    -   F is selected from the group comprising Ser, Gly, and Thr;    -   G is selected from the group comprising Leu, Val, and Phe; and    -   H is selected from the group comprising Ile, Ser, Leu, Val, Phe,        and Ala.

In one embodiment, the present invention contemplates a peptidederivative having the general formula R₁-[Arg and/orLys]_(n=0-4)-A-B-C-D-E-F-G-H-[Arg and/or Lys]_(n=0-4)-R₂ (SEQ ID NO:15)or a disulfide-bridged, linear dimer thereof, or a cyclic dimer thereof,wherein:

A may be i) absent; ii) 1-7 amino acids selected from the groupincluding, but not limited to, Gln, Ser, Gly, Tyr, Cys, Thr, Asn, Lys,Arg, Lys, or Gln, or iii) 1-7 amino acids selected from the groupincluding, but not limited to, Pro, Phe, Leu, Ala, Val, Ile, Met, Arg,Lys, or Trp;

B may be 1-3 amino acids selected from the group including, but notlimited to, Ser, Gly, Tyr, Cys, Thr, Asn, Lys, Arg, Lys, or Gln;

C may be 1-2 amino acids selected from the group including, but notlimited to, Pro, Phe, Leu, Ala, Val, Ile, Met, Trp, or Cys;

D may be selected from the group including, but not limited to, Gln,Ser, Gly, Tyr, Cys, Ile, and Asn;

E may be selected from the group including, but not limited to, Arg,Lys, and His;

F may be selected from the group including, but not limited to, Gln,Ser, Gly, Tyr, Cys, Ile, and Asn;

G may be 1-4 amino acids selected from the group including, but notlimited to, Pro, Phe, Leu, Ala, Val, Ile, Met, Trp, Arg, or Lys;

H may be i) absent; ii) 1-7 amino acids selected from the groupincluding, but not limited to, Gln, Ser, Gly, Tyr, Cys, Thr, Asn, Lys,Arg, Lys, or Gln, or iii) 1-7 amino acids selected from the groupincluding, but not limited to, Pro, Phe, Leu, Ala, Val, Ile, Met, Arg,Lys, or Trp;

R₁ and R₂ may be either i) absent; a conjugated lipid selected from thegroup including, but not limited to, Gly-Tris-monopalmitate,-dipalmitate and -tripalmitate; or a conjugated sugar selected from thegroup including, but not limited to, 1-amino-glucose succinate,2-aminododecanoate, or myristoylate. See, FIG. 8.

As referred to herein, hydrophobic amino acids include, but are notlimited to, Ala, Val, Leu, Ile, Pro, Phe, Trp, and Met; positivelycharged amino acids include, but are not limited to, Lys, Arg and His;and negatively charged amino acids include, but are not limited to, Aspand Glu.

The general formula above represents one embodiment of a TREM-1transmembrane segment comprising at least one conserved domain thatcontains highly homologous sequences between species. In one embodiment,a TREM-1 transmembrane segment comprises GFLSKSLVF(Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe; human amino acid residues 213-221;Accession No. Q9NP99) (SEQ ID NO: 16) along with various lipid and/orsugar derivatives that may, or may not, have a disulfide bridged dimer.In another embodiment, a TREM-1 transmembrane segment comprisesKKILLAGGFLSKSLVRSVLFAKR (SEQ ID NO: 17), wherein said sequence meets thecriteria for the above outlined general formula.

In one embodiment, the present invention contemplates a method ofrational designing of the peptides and lipid- and/or sugar-conjugatedpeptides consisting of L- or D-stereoisomeric amino acids in order toincrease effectiveness of the peptides in inhibiting the function of aTREM-1/DAP-12 receptor complex. In one embodiment, the method comprisessubstituting at least one amino acid of a TREM-1 transmembrane corepeptide (i.e., for example, and arginine or a lysine into at least onealpha-helix of the Peptide Core and/or Extended Peptide), therebyincreasing binding to the transmembrane domain of DAP-12 chain. See,FIG. 7.

In another embodiment, the method comprises conjugating at least onelipid and/or at least one sugar to the C- and/or N-termini of thepeptide, thereby increasing binding to the transmembrane domain of theDAP-12 chain and/or improving the penetration of the peptide variantinto the cell membrane. In one embodiment, the lipid- and/orsugar-conjugated peptide variants comprise D-amino acids, therebyincreasing resistance to protease degradation. In one embodiment, aprotease resistant peptide variant is useful clinically for inhibitingTREM-mediated cell activation in myeloid cell-mediated disorders.

In some embodiments, conjugated peptide variants are synthesized usingthe standard procedures (Amon et al. Biochim Biophys Acta 2006;1763:879-88; Whittaker et al. Pept Res 1993; 6:125-8; In: Chemistry ofPeptide Synthesis, N. Leo Benoiton (ed.), CRC, 2005; Gerber et al. FasebJ 2005; 19:1190-2; Kliger et al. J Biol Chem 1997; 272:13496-505;Merrifield et al. Biochemistry 1982; 21:5020-31).

In one embodiment, the rational design method comprises inserting atleast one polyarginine and/or polylysine sequence into a TREM-1transmembrane sequence, thereby increasing binding to a transmembranedomain of an DAP-12 chain and/or improving the penetration of thepeptide variant into the cell membrane. Other modifications of thepeptides contemplated herein include, but are not limited to,modifications to side chains, incorporation of unnatural amino acidsand/or their derivatives during peptide synthesis and the use ofcrosslinkers and other methods which impose conformational constraintson the peptides. It may also be possible to add various groups to thepeptide of the present invention to confer advantages such as increasedpotency or extended half life in vivo without substantially decreasingthe biological activity of the peptide. It is intended that suchmodifications to the peptide of the present invention which do notresult in a decrease in biological activity are within the scope of thepresent invention.

Any combination of the above embodiments may be used together in orderto increase effectiveness of the peptide variants to inhibit thefunction of a TREM-1/DAP-12 receptor complex. The most effectiveinhibitory peptides and derivatives thereof may be identified by typicalscreening assay procedures for evaluation of inhibition of TREM-mediatedcell activation and function (Ford J. W. & McVicar D. W. Curr OpinImmunol 2009; 21, 38-46; Klesney-Tait et al. Nat Immunol 2006;7:1266-73).

5. Peptide-Based Inhibitors

Although it is not necessary to understand the mechanism of aninvention, it is believed that inhibition of a TREM receptor (i.e., forexample, TREM-I/DAP-12 receptor complex) signaling can be achieved bybinding of a peptide-based inhibitor to the transmembrane (TM) domain ofthe DAP-12 chain, thus substituting the TREM-1 subunit and abolishingthe interaction between the TM domains of the TREM-1 and DAP-12 chains.One possible result is the inhibition of TREM-1-mediated transmembranesignaling because ligand binding to the extracellular domain of a TREM-1ligand recognition subunit does not lead to DAP-12 signaling subunitclustering (i.e., for example, oligomerization). It is further believedthat this clustering induces the phosphorylation of tyrosine residues inthe intracellular DAP-12 domain and initiates downstream signaling.

The TM domains of TREM-1 and DAP-12 subunits comprise hydrophobicsequences that may adopt a stable alpha-helical structure within a cellplasma membrane lipid bilayer. It is hypothesized that electrostaticinteractions between these TM domains maintain the integrity of theTREM-1/DAP-12 receptor signaling complex and are provided by theinteraction between positively charged Lys residue of the TREM-1 TMdomain and two negatively charged Asp residues of the TM domains of theDAP-12 dimer.

It might be suggested that, theoretically, the simplest and the mostselective and effective peptide inhibitor would be a synthetic peptidecorresponding to the TM domain of TREM-1 subunit. However, as severalembodiments of the present invention contemplate, peptide inhibitorsequence, alone, is not the only relevant consideration. In oneembodiment, a peptide inhibitor targeted to the transmembraneinteractions should be optimized for cell membrane binding. In oneembodiment, a peptide inhibitor should be optimized for membraneinsertion, thereby attaining a close spatial proximity and/or properorientation to an interacting partner (i.e., for example, a TM domain ofa DAP-12 dimer). In one embodiment, a peptide inhibitor should beoptimized for binding effectiveness to an interacting partner.

Although it is not necessary to understand the mechanism of aninvention, it is believed that a peptide inhibitor comprising the wildtype TM domain of TREM-1 is not optimized for each of the above threefactors. Other embodiments, however, are contemplated by the presentinvention by using extracellularly administered synthetic peptides whichare optimized for at least one of the above three considerations. Thus,the inhibition of cell activation by the TREM-1 core peptide can besignificantly improved in terms of efficiency by rational design of thepeptide-based inhibitors. For example, the inhibition activity the Tcell antigen receptor core peptide has been reported to increase from 30to 80% by lipidation of the relevant peptide inhibitors.

In summary, the present invention contemplates optimizing theeffectiveness and selectivity of peptide inhibitors forTREM-1/DAP-12-mediated signaling, by adhering to at least one of theseguidelines:

1) ability to effectively bind to the cell plasma membrane and insertinto the membrane (i.e., for example, increasing hydrophobicity);

2) ability to adopt helical conformation upon membrane binding andinsertion (i.e., for example, increasing intrinsic helicity);

3) ability to selectively and effectively bind to the TM domain of theDAP-12 subunit, thus effectively competing with the TREM-1 subunit forthe binding to the DAP-12 subunit (i.e., for example, by increasingstable alpha-helixes).

The following guidelines were used to develop a method of rationaldesigning of the peptides in order to increase effectiveness of thepeptides in inhibition of function of the TREM-1/DAP-12 receptorcomplex.

5.1. Hydrophobicity

The hydrophobicity (or lipophilicity) of peptides and peptide analoguesmay be increased by i) inserting hydrophobic regions; ii) improving theability of a peptide-based inhibitors to bind the membrane; and/or iii)improving the ability of a peptide-based inhibitor to insert into amembrane. In one embodiment, hydrophobic regions may be inserted into apeptide inhibitor sequence by using lipophilic groups including, but notlimited to, myristoylate-, 1-amino-glucose succinate,2-aminododecanoate, or Gly-Tris-palmitate, -dipalmitate or-tripalmitate, coupled to the N- and/or C-termini of a peptide. In oneembodiment, the membrane binding/insertion ability of a peptideinhibitor may be improved by using highly positively charged poly-Lys orpoly-Arg sequences coupled to an N- and/or C-terminus. A general formulasummarizing many suggested inhibitory peptides and/or compositions ispresented that incorporates both approaches that are expected toincrease the effectiveness of the peptides to inhibit the function of aTREM-1/DAP-12 receptor complex. See, FIG. 8.

Lipid-binding activity of the test peptide-based inhibitors can bepredicted using ProtParam™ software (us.expasy.org/tools/protparam.html)and experimentally evaluated by different techniques such as, forexample, surface plasmon resonance (SPR) or sucrose-loaded vesiclebinding assay. Based on the obtained results, a peptide-based inhibitorwith optimal membrane-binding activity can be chosen. For example,“Grand Average Of Hydropathy” (GRAVY) scores for peptides can beobtained using ProtParam™, in which a score >-0.4 (=mean score forcytosolic proteins) indicates the probability for membrane association(i.e., for example, the higher the score, the greater the probabilityfor membrane association).

5.2. Helicity

As discussed above, the primary sequence of the parent inhibitorypeptide, the TREM-1 core peptide, can be modified to improve the abilityof various peptide-based inhibitors contemplated herein to adopt helicalconformation upon membrane binding and insertion. See, FIG. 8. Overallprotein folding may be specified by hydrophobic-polar residuepatterning, whereas the bundle oligomerization state, detailedmain-chain conformation, and interior side-chain rotamers may beengineered by computational enumerations of packing in alternatebackbone structures. Main-chain flexibility is incorporated through analgebraic parameterization of the backbone (Harbury et al. Science 1998;282:1462-7).

Peptide helicity of the designed primary sequences of variouspeptide-based inhibitors contemplated herein can be first evaluatedcomputationally using secondary structure prediction programs. (i.e.,for example, Expasy Proteomics Tools; expasy.org/tools). The mostpromising inhibitors can be measured experimentally for intrinsic and/orinduced helicity using circular dichroism (CD) spectroscopy. CDspectroscopy is used to analyze the secondary structure of a proteinand/or peptide. Specifically, CD spectroscopy measures differences inthe absorption of left-handed polarized light versus right-handedpolarized light which arise due to structural asymmetry. The absence ofregular structure results in zero CD intensity, while an orderedstructure results in a spectrum which can contain both positive andnegative signals. alpha-helix, beta-sheet, and random coil structureseach give rise to a characteristic shape and magnitude of CD spectrum.The approximate fraction of each secondary structure type that ispresent in any peptide or protein can thus be determined by analyzingits far-UV CD spectrum as a sum of fractional multiples of suchreference spectra for each structural type. Like all spectroscopictechniques, the CD signal reflects an average of the entire molecularpopulation. Thus, while CD can determine that a peptide or proteincontains about 50% alpha-helix, it cannot determine which specificresidues are involved in the alpha-helical portion. Based on theobtained results, a peptide-based inhibitor optimized with the predictedand/or observed, intrinsic and/or induced optimal helicity can bechosen.

Alternatively, secondary structure prediction programs (for example,expasy.org/tools/) may be used to accurately predict the peptidehelicity based on primary sequence of the computationally designedpeptide-based inhibitors. A few of the available programs include, butare not limited to: a) AGADIR—An algorithm to predict the helicalcontent of peptides; b) APSSP—Advanced Protein Secondary StructurePrediction Server; c) GOR—Gamier et al. Methods Enzymol 1996;266:540-53; d) HNN—Hierarchical Neural Network method (Guermeur et al.Bioinformatics 1999; 15:413-21) e) Jpred—A consensus method for proteinsecondary structure prediction at University of Dundee; f) JUFO—Proteinsecondary structure prediction from sequence (neural network); g)nnPredict—University of California at San Francisco (UCSF); h)Porter—University College Dublin; i) PredictProtein—PHDsec, PHDacc,PHDhtm, PHDtopology, PHDthreader, MaxHom, EvalSec from ColumbiaUniversity; j) Prof—Cascaded Multiple Classifiers for SecondaryStructure Prediction; k) PSA—BioMolecular Engineering Research Center(BMERC)/Boston; l) PSIpred—Various protein structure prediction methodsat Brunel University; m) SOPMA—Geourjon and Deleage, 1995; n)Sspro—Secondary structure prediction using bidirectional recurrentneural networks at University of California; and o) DLP—Domain linkerprediction at RIKEN.

5.3. Alpha-Helix Stability

Although it is not necessary to understand the mechanism of aninvention, it is believed that the TM domains of the TREM-1 and theDAP-12 subunits represent stable alpha-helixes and, thus, theinteractions can be presented using helix-wheel diagrams. See, FIGS. 5,6, and 7. As described in (US Pat Appl 20090075899) and incorporatedherein by reference in its entirety, these diagrams are based on theprimary peptide/protein sequence and can be created using commerciallyand publicly available programs (i.e., including, but not limited to,Antheprot v. 6.0; antheprot-pbil.ibcp.fr/; or Helical Wheel CustomImages and Interactive Java Applets;cti.itc.virginia.edu/.about.cmg/Demo/wheel/wheelApp.html). Thesediagrams can be used for evaluation of close proximity and/or properorientation of positively charged amino acid residue(s) of the peptideor peptide analogue of interest towards an interacting partner (i.e.,for example, negatively charged TM residues of a DAP-12 dimer).

As shown in the FIG. 5, the electrostatic interaction between the TREM-1TM positively charged Lys (K, blue) and the negatively charged asparticacid pair (D, pink) in the DAP-12 dimer stabilize the association ofthese respective subunits, thereby playing a role in ligand-inducedTREM-1-mediated cell activation and response. Some embodiments ofpeptide-based inhibitors contemplated by the present invention aim tointerrupt this interaction and replace the TREM-1 subunit of theTREM-1/DAP-12 receptor complex. In one embodiment, peptide-basedinhibitors can be computationally designed to increase theircompetitiveness with the TREM-1 subunit. In one embodiment,competitiveness may be increased by using a conservative amino acidsubstitution of lysine for arginine. In another embodiment,competitiveness may be increased by inserting a positively charged aminoacid residue (i.e., for example, arginine and/or lysine). In oneembodiment, the insertion and/or substitution is located within analpha-helix of the peptide-based inhibitors (i.e., for example, a TREM-1core or Extended Peptides; FIG. 7), thereby increasing the bindingactivity to transmembrane domains of the DAP-12 signaling subunit dimerand enhancing the effectiveness of the peptides to inhibit the functionof the a TREM-1/DAP-12 receptor complex.

6. Peptide-Based Inhibitor Sequence Listings

A list of the sequences of the peptides and peptide analogues shownbelow includes, but is not limited to, peptide-based inhibitorspredicted to be effective in inhibiting the TREM-1/DAP-12 signalingmechanism. See Table 2.

Accordingly, it is intended that the present invention includes withinits scope peptides which include additional amino acids to the “core”sequence of the peptide of the present invention and which affect thetransmembrane interactions between the TREM-1 subunit and DAP-12 dimer.

TABLE 2  Exemplary Peptide-Based TREM-1/DAP-12 Complex Inhibitor Sequences Sequence (the “core”sequence of the peptide of   ## R₁ ^(a)the present invention is underlined) R₂ ^(b)  1 —IVILLAGGFLSKSLVFSVLFA (parent) (SEQ ID NO: 18) − (TREM-1 TM peptide)  2— GFLSKSLVF (SEQ ID NO: 19) − (TREM-1 TM core peptide)  3 —IVILLAGGFLSKSLVFSVLFA (SEQ ID NO: 20) +  4 LA IVILLAGGFLSKSLVFSVLFA(SEQ ID NO: 21) −  5 Myr GSVILLAGGFLSKSLVFSVLFA (SEQ ID NO: 22) −  6 LAIVILLAGGFLSKSLVFSVLFA (SEQ ID NO: 23) +  7 — KKILLAGGFLSKSLVFSVLFAKR(SEQ ID NO: 24) −  8 — KKILLAGGFLSKSLVFSVLIFAKR (SEQ ID NO: 25) +  9 —(IVILLAGGFLSKSLVFSVLFA)₂ ^(c) (SEQ ID NO: 26) − 10 —IVILLACGFLSKSLVFSVLFA (SEQ ID NO: 27) − 11 — (IVILLAC*GFLSKSLVFSVLFA)₂^(d) (SEQ ID NO: 28) − 12 — IVILLAGGFLSKSLVRSVLFA (SEQ ID NO: 29) − 13 —IVILLAGGFLSKSLVRSVLFA (SEQ ID NO: 30) + 14 LA IVILLAGGFLSKSLVRSVLFA(SEQ ID NO: 31) − 15 Myr GSILLAGGFLSKSLVRSVLFA (SEQ ID NO: 32) − 16 —KKILLAGGFLSKSLVRSVLFAKR (SEQ ID NO: 33) − 17 LA KKILLAGGFLSKSLVRSVLFAKR(SEQ ID NO: 34) − 18 — KKILLAGGFLSKSLVRSVLFAKR (SEQ ID NO: 35) + 19 —(IVILLAGGFLSKSLVRSVLFA)₂ (SEQ ID NO: 36) − 20 — IVILLACGFLSKSLVRSVLFA(SEQ ID NO: 37) − 21 — (IVILLAC*GFLSKSLVRSVLFA)₂ (SEQ ID NO: 38) − 22 —IVILLAGRFLSKSLVRSVLFA (SEQ ID NO: 39) − 23 LA IVILLAGRFLSKSLVRSVLFA(SEQ ID NO: 40) − 24 — IVILLAGRFLSKSLVRSVLFA (SEQ ID NO: 41) + 25 —KKILLAGRFLSKSLVRSVLFAKR (SEQ ID NO: 42) − 26 LA KKILLAGRFLSKSLVRSVLFAKR(SEQ ID NO: 43) − 27 — KKILLAGRFLSKSLVRSVLFAKR (SEQ ID NO: 44) + 28 —(IVILLAGRFLSKSLVRSVLFA)₂ (SEQ ID NO: 45) − 29 — IVILLACRFLSKSLVRSVLFA(SEQ ID NO: 46) − 30 — (IVILLAC*RFLSKSLVRSVLFA)₂ (SEQ ID NO: 47) − 31 —GFLSKSLVF (SEQ ID NO: 48) − 32 LA GFLSKSLVF (SEQ ID NO: 49) − 33 —GFLSKSLVF (SEQ ID NO: 50) + 34 — (GFLSKSLVF)₂ (SEQ ID NO: 51) − 35 —ACGFLSKSLVF (SEQ ID NO: 52) − 36 — (AC*GFLSKSLVF)₂ (SEQ ID NO: 53) − 37— GLLSKSLVF (SEQ ID NO: 54) − 38 LA GLLSKSLVF (SEQ ID NO: 55) − 39 —GLLSKSLVF (SEQ ID NO: 56) + 40 — (GLLSKSLVF)₂ (SEQ ID NO: 57) − 41 —ACGLLSKSLVF (SEQ ID NO: 58) − 42 — (AC*GLLSKSLVF)₂ (SEQ ID NO: 59) − 43— GLLSKTLVF (SEQ ID NO: 60) − 44 LA GLLSKTLVF (SEQ ID NO: 61) − 45 —GLLSKTLVF (SEQ ID NO: 62) + 46 — (GLLSKTLVF)₂ (SEQ ID NO: 63) − 47 —ACGLLSKTLVF (SEQ ID NO: 64) − 48 — (AC*GLLSKTLVF)₂ (SEQ ID NO: 65) − 49— GFLSKSLVR (SEQ ID NO: 66) − 50 LA GFLSKSLVR (SEQ ID NO: 67) − 51 —GFLSKSLVR SEQ ID NO: 68 + 52 — (GFLSKSLVR)₂ SEQ ID NO: 69 − 53 —ACGFLSKSLVR SEQ ID NO: 70 − 54 — (AC*GFLSKSLVR)₂ SEQ ID NO: 71 − 55 —KFLSKSLVR SEQ ID NO: 72 − 56 LA KFLSKSLVR SEQ ID NO: 73 − 57 — KFLSKSLVRSEQ ID NO: 74 + 58 — (KFLSKSLVR)₂ SEQ ID NO: 75 − 59 — ACKFLSKSLVRSEQ ID NO: 76 − 60 — (AC*RFLSKSLVR)₂ SEQ ID NO: 77 − 61 —VTISVICGLLSKSLVFIILFI SEQ ID NO: 78 − 62 — (VTISVICGLLSKSLVFIILFI)₂SEQ ID NO: 79 − 63 — (VTISVIC*GLLSKSLVFIILFI)₂ SEQ ID NO: 80 − 64 —IIIPAACGLLSKTLVFIGLFA SEQ ID NO: 81 − 65 — (IIIPAACGLLSKTLVFIGLFA)₂SEQ ID NO: 82 − 66 — (IIIPAAC*GLLSKTLVFIGLFA)₂ SEQ ID NO: 83 − 67 —ILPAVCGLLSKSLVFIVLFVV SEQ ID NO: 84 − 68 — ILPAVCKLLSKSLVFIVLFVVSEQ ID NO: 85 − 69 — (ILPAVCGLLSKSLVFIVLFVV)₂ SEQ ID NO: 86 − 70 —(ILPAVC*GLLSKSLVFIVLFVV)₂ SEQ ID NO: 87 ^(a)N-terminal group: LA,lipoamino acid, 2-aminododecanoate; Myr, myristoylate. ^(b)C-terminalgroup: Gly-Tris-tripalmitate. ^(c)Cyclic peptide. ^(d)Disulfide-linkeddimer (or disulfide-linked cyclic dimer). *Cys involved in disulfidebond formation. Abbreviations: TM, transmembrane

Abbreviations: TM, Transmembrane

7. Peptide Variant Consensus Sequences

Based upon the specific sequences contemplated in Table 2, the followingconsensus sequences may be constructed:

SEQ ID NO: 1: G-X₁-X₂-L-S-X₃-X₄-L-V-X₅-X₆-X₇-X₈-X₉-X₁₀, wherein X₁consists of G, C or is selected from the group consisting of R, K or H;X₂ is selected from the group consisting of L, F or I; X₃ is selectedfrom the group consisting of R, K or H; X₄ is selected from the groupconsisting of S or T; X₅ consists of F or is selected from the groupconsisting of R, K or H; X₆ consists of S, I, L or nothing; X₇ consistsof V, I, L, G or nothing; and X₈, X₉, and X₁₀ consist of L, F, A ornothing.

SEQ ID NO: 2: X₁-X₂-X₃-G-F-L-S-K-S-L-V-R-V-X₄-X₅, wherein X₁ consists ofG, C or nothing; and X₂, X₃, X₄, and X₅ consist of K, R, or nothing.

SEQ ID NO: 3:X₁-X₂-X₃-L-X₄-X₅-X₆-X₇-G-X₈-L-S-K-X₉-L-V-F-X₁₀-X₁₁-L-F-X₁₂-X₁₃-X₁₄-X₁₅,wherein X₁ consists of G or nothing; and X₂, X₃, X₁₄, and X₁₅ consist ofK, R, or nothing; X₄, X₅, X₆, and X₇ consist of P, A, V, C, L, I, S, Gor nothing; X₈ consists of F, L or I; X₉ consists of S or T; X₁₀, X₁₁,X₁₂, and X₁₃ consist of S, I, L, G, V, A, or nothing.

8. Therapeutic Applications of TREM-1/DAP-12 Peptide Inhibitors

The invention further provides clinically therapeutic methods ofintervening and modulating TREM-1 function comprising using an agentselected from the group of agents or compositions of the presentinvention that block/inhibit/prevent/disrupt interactions between theTREM-1 chain and the homodimeric DAP-12 subunit of the TREM-1/DAP-12complex.

High-throughput screening methods that can be used for screening andoptimizing the effective TREM-1 peptide inhibitors of the presentinvention that block/inhibit/prevent/disrupt interactions between theTREM-1 chain and the homodimeric DAP-12 subunit of the TREM-1/DAP-12receptor complex are described in US Pat Appl 20090075899 andincorporated herein by reference in its entirety. See also FIGS. 2, 4,9, 10, and 11.

Various therapeutic applications of TREM-1 inhibitors are described in(Bouchon et al. J Immunol 2000; 164:4991-5; Ford J. W. & McVicar D. W.Curr Opin Immunol 2009; 21, 38-46; Bouchon et al. Nature 2001;410:1103-7; Gibot S. Crit Care 2005; 9:485-9; Gibot et al. J Exp Med2004; 200:1419-26; Gibot et al. Shock 2009; 32:633-7; Gibot et al. CritCare Med 2008; 36:504-10; Klesney-Tait et al. Nat Immunol 2006;7:1266-73; Murakami et al. Arthritis Rheum 2009; 60:1615-23; Sharif 0. &Knapp S. Immunobiology 2008; 213:701-13; US Pat Appls 20080247955,20060246082, 20090081199, and 20030165875; U.S. Pat. No. 6,420,526; Linget al. Chinese Med J 2010; 123:1561-5; Ho et al. Am J Respir Crit CareMed 2008; 177:763-70) and incorporated herein by reference in theirentirety.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

The following non-limiting Examples are put forth so as to provide thoseof ordinary skill in the art with illustrative embodiments as to how thecompounds, compositions, articles, devices, and/or methods claimedherein are made and evaluated. The Examples are intended to be purelyexemplary of the invention and are not intended to limit the scope ofwhat the inventor regard as his invention. Efforts have been made toensure accuracy with respect to numbers (e.g., amounts, temperature,etc.) but some errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is in ° C.,or is at ambient temperature, and pressure is at or near atmospheric.

Example 1: Synthesis of Peptides

This example demonstrates one embodiment of a synthesized TREM-1transmembrane peptide.

The first step is to synthesize the short hydrophobic peptidecorresponding to a portion of a TREM-1 transmembrane domain sequence.Although it is not necessary to understand the mechanism of aninvention, it is believed that this peptide affects the TREM-1/DAP-12receptor complex assembly and may interact with the DAP-12 homodimer ina competitive fashion.

The synthesis of peptides may involve the use of protecting groups.Peptides can be synthesized by linking an amino group to a carboxylgroup that has been activated by reaction with a coupling agent, such asdicyclohexylcarbodiimide (DCC). The attack of a free amino group on theactivated carboxyl leads to the formation of a peptide bond and therelease of dicyclohexylurea. It can be necessary to protect potentiallyreactive groups other than the amino and carboxyl groups intended toreact. For example, the α-amino group of the component containing theactivated carboxyl group can be blocked with a tertbutyloxycarbonylgroup. This protecting group can be subsequently removed by exposing thepeptide to dilute acid, which leaves peptide bonds intact.

In one embodiment, the amino acid sequence of a competitive peptidecomprises NH₂-Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe-OH (i.e., GFLSKSLVF)(SEQ ID NO: 16), hereafter referred to as “core peptide” or “CP”. Inanother embodiment, the amino acid sequence of a competitive peptidecomprises NH₂-Gly-Phe-Leu-Ser-Ala-Ser-Leu-Val-Phe-OH (i.e., GFLSASLVF)(SEQ ID NO: 88) wherein, Lys⁵ of CP substituted with Ala⁵, hereafterreferred to as “CP-A”.

Although it is not necessary to understand the mechanism of aninvention, it is believed that the positively charged Lys₅ in the CPtransmembrane domain of TREM-1 forms a salt bridge to an aspartic acidresidue in the transmembrane domain of the DAP-12 chain. Thus, CP-A maybe considered as “control peptide” because of the Lys⁵ substitution. Inone embodiment, a scrambled peptide containing the same amino acids as“core peptide” but in a totally different sequence order (e.g.,LFGFLVSSK) (SEQ ID NO: 102) can be similarly synthesized and used ascontrol peptide.

Unprotected peptides can be purchased from specialized companies (i.e.,Sigma-Genosys, Woodlands, Tex., USA) with greater than 95% purity asassessed by HPLC. Peptide molecular mass can be checked bymatrix-assisted laser desorption ionization mass spectrometry.

Example 2: Solubility

This example demonstrates that the hydrophobic properties of CP peptidesand other peptides and compositions of the present invention may beovercome without risking cell toxicity. This example furtherdemonstrates that the hydrophobic properties of TREM-1 core peptides maybe overcome without risking cell toxicity.

The CP and CP-A peptides can be hydrophobic and insoluble in aqueoussolutions. A variety of solvents and carriers can be tested to improvetheir solubility. Solvents and/or carriers that improve solubility of CPand CP-A include, but are not limited to, ethanol, dimethylsulphoxide(DMSO), dimethyl formamide (DMF), and trifluoracetic acid (TFA). Whenusing DMSO as a solvent, the final concentration used in the cellfunction experiments may range from 0.063%-0.250%. Stock solutions of CPand CP-A can be prepared in DMSO and used at a 1:2000, 1:1000, or 1:400dilution. In one embodiment, drug delivery systems (i.e., for example,lipid vesicles) can be used to increase solubility and bioavailabilityof CP and CP-A.

Example 3: Preparation of Small Unilamellar Vesicles (SUVs)

Model membranes composed of zwitterionic and anionic phospholipids andtheir mixtures in proportion similar to that found in vertebrate cellmembranes can be produced as follows.Dimyristoyl-L-alpha-phosphatidylcholine (DMPC) anddimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG) are dissolved in drychloroform and chloroform/methanol (2:1), respectively, to give 10 mg/mlsolutions. These are evaporated under reduced pressure and the resultinglipid films are vacuum-dried overnight. Lipids are hydrated byresuspending in HEPES buffer for 60 min at 34° C. to give 0.5 mMconcentration in respect of phospholipids. The solution is sonicated inan ultrasonic bath for 20 min. Eight cycles of freeze/thawing arefollowed by extrusion through polycarbonate filters, first 100 nm (21times), then 50-nm pore diameters (21 times), using a Lipofast apparatus(Avestin, Ottawa, Canada) and the SUVs are used immediately.

Example 4: Surface Plasmon Resonance (SPR) Analysis of Lipid BindingActivity

SPR is carried out on a BIAcore™ 2000 instrument using Pioneer SensorChip L1 and HEPES (HBS-N, Biacore) as running buffer. The chip surfaceis cleaned with 40 mM octyl glycoside (30 mL, 10 mL/min) followed byrunning buffer (100 mL, 10 mL/min). Liposomes (small unilamellarvesicles, SUVs), made in accordance to Example 7, are injected (100 mL,5 mL/min) giving a response of about 8000 RU. 10 mM NaOH (40 mL, 10mL/min) removes any multilamellar vesicles from the surface that isfollowed by 10 mM glycine, pH 2.2 (10 mL, 10 mL/min), before injecting aTREM-1 peptide or peptide analogue inhibitor (100 mL, 5 mL/min). Afterpeptide injection the dissociation stage is 1200 s. Regeneration of thesensor chip is achieved with 40 mM octyl glycoside (30 mL, 10 mL/min).All SPR experiments are run at 25° C. and all analyses are performedusing BIAevaluation software (Biacore). For the comparative binding of apeptide and/or peptide analogue, a determination of percentage bindingis expressed as a percentage of the wild-type peptide binding. Thevalues are representative of three injections under identical conditionsfor each peptide. To study the effect of interaction time on thewild-type peptide binding, the “variable contact times” injectioncommand is utilized on the Biacore™ 2000 instrument. This is achieved byswitching additional flow cells into the flow path as the injectionproceeds; thus the injections end at the same time.

Example 5: Preparation of Sucrose-Loaded Vesicles

This example describes the preparation of sucrose-loaded largeunilamellar vesicles (LUVs) for use in a sucrose-loaded vesicle bindingassay. The corresponding lipid, the zwitterionic lipid1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or the acidiclipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), or lipidmixtures in CHCl₃ are evaporated under argon and then vacuum-dried for 3h at 20° C. The dried lipid is resuspended in 176 mM sucrose and 1 mMMOPS at pH 7.0; the air is displaced with argon. LUVs are made afterfive freeze-thaw cycles by extruding multilamellar vesicles 10 timesthrough a stack of two polycarbonate filters (100-nm pore diameter) inan Avanti mini-extruder (Avanti Polar Lipids, Alabaster, Ala.). Thevesicle solution is diluted 5 times with 1 mM MOPS buffer, pH 7.0,containing 0.1 M KCl and osmotic to the internal sucrose buffer, andcentrifuged at 100,000 g for 1 h at 25° C. using a table-top BeckmanTL-100 ultracentrifuge equipped with a TLA-45 rotor. The supernatant isremoved, and the lipid pellet is resuspended in the same buffersolution. The final concentration of the vesicle solution is determinedusing a phosphorous assay.

Example 6: Sucrose-Loaded Vesicle Binding Assay

In the membrane-binding assay, peptide or peptide analogue in a finalconcentration of 10 mM is mixed with the sucrose-loaded LUVs;[peptide]<<[lipid] so that the peptide does not bind a significantfraction of the lipid. After 15 min of equilibration at room temperature(20° C.), vesicle-bound peptide or peptide analogue is separated bycentrifugation (for 1 h at 100,000 g and 25° C.). Ninety percent of thesupernatant and pellet is evaluated for protein content using afluorescamine assay. The percentage of the protein bound at a givenlipid concentration is calculated and corrected for the 1-3% lipid thatremains in the supernatant.

Example 7: Circular Dicroism Spectroscopy Analysis of a PeptideSecondary Structure

Far-UV CD spectra can be recorded on an Aviv 202 spectropolarimeter(AVIV Instruments, Lakewood, N.J.) with 0.01 mM peptide or peptideanalogue in the absence or presence of SDS and/or lipids, or helicityinducers, such as TFE, in phosphate buffered saline buffer (PBS; 137 mMNaCl, 10 mM sodium phosphate, 2.7 mM KCl, pH 7.4) in 1.0 mm path-lengthcells. Data are collected at 25° C. every nanometer from 260 to 190 nmwith 1.0 s averaging per point and a 1 nm bandwidth. The CD spectra ofat least six scans are signal averaged, baseline corrected bysubtracting an averaged buffer spectrum, and normalized to molar residueellipticity.

Example 8: Protection of Mice from Death by Septic Shock with TREM-1Peptide Variants

The experiment can be conducted analogously to that described in US PatAppl 20060246082, which is incorporated herein by reference in itsentirety.

In the experiments of this example, the peptides are administered in avolume of 200 ul. To assess the ability of TREM-1 transmembrane peptidesto protect mice from LPS-induced endotoxaemia, the CP and CP-A peptidesat various concentrations are administered 1 hour before a lethal doseof lipopolysaccharide (LPS). Lethality is monitored over time andcompared with animals that have received control injections of vehiclealone.

In order to investigate whether treatment with TREM-1 TM peptidevariants can be delayed until after the administration of LPS, thepeptides are injected at various time points after LPS injection. Thepeptides can be further investigated for their ability to protectagainst septic shock in another widely used experimental model ofsepsis, the “CLP” model (Cecal Ligation and Puncture).

Example 9: Studies on the Modulation of the Inflammatory Response inMurine Sepsis by TREM-1 Peptide Variants

The experiments can be conducted analogously to that described in US PatAppl 20060246082, which is incorporated herein by reference in itsentirety.

Methods

Preparation of Monocytes from Peripheral Blood

Ten mL of peripheral blood samples are collected on EDTA-K from healthyvolunteer donors. After dilution in RPMI (Life Technologies, GrandIsland, N.Y.), blood is centrifuged for 30 min at room temperature overa Ficoll gradient (Amersham Pharmacia, Uppsala, Sweden) to isolate PBMC.The cells recovered above the gradient are washed and counted. In orderto deplete the suspensions of lymphocytes, cells are then plated in24-well flat-bottom tissue culture plates (Corning, Corning, N.Y.) at aconcentration of 5×10⁶/mL and allowed to adhere during 2 hours at 37° C.The resulting lymphocyte suspension is discarded and the adheringmonocytic cells are maintained in a 5% CO₂ incubator at 37° C. incomplete medium (RPMI 1640, 0.1 mM sodium pyruvate, 2 mM Penicillin, 50ug/mL Streptomycin; Life Technologies) supplemented with 10% FCS(Invitrogen, Cergy, France).

In Vitro Stimulation of Monocytes

For activation, monocytes are cultured in the presence of E. coli LPS(O111:B4, 1 ug/mL, Sigma-Aldrich, La Verpilliere, France). Cellviability is assessed by trypan blue exclusion and by measuring lactatedehydrogenase release. In some experiments, this stimulus is given incombination with TNF-alpha (5 to 100 ng/mL, R&D Systems, Lille, France),IL-1beta (5 to 100 ng/mL, R&D Systems), rIFN-gamma (up to 100 U/mL, R&DSystems), rIL-10 (500 U/ml, R&D Systems) or up to 100 ng/mL of CP orCP-A.

In order to activate monocytes through TREM-1, an anti-TREM-1 agonistmonoclonal antibody (R&D Systems) is added as follows: flat-bottomplates are precoated with 10 ug/mL anti-TREM-1 per well. After thoroughwashing in phosphate buffered saline (PBS), the monocyte suspensions areadded at a similar concentration as above. Some experiments areperformed in the presence of protease inhibitors (PMSF and ProteaseCocktail Inhibitor; Invitrogen). Cell-free supernatants are assayed forthe production of TNF-alpha and IL-1beta by ELISA according to therecommendations of the manufacturer (BD Biosciences, San Diego, USA). Toaddress the effect of CP on NF-κB activity in monocytes, an ELISA-basedassay is performed (BD Mercury Transfactor Kit, BD Biosciences).Monocytes are cultured for 24 hours in the presence of E. coli LPS(O111:B4, 1 ug/mL), and/or an agonist anti-TREM-1 monoclonal antibody(10 ug/mL), with or without TREM-1 CP or control peptide added atvarious concentrations. Whole-cell extracts are then prepared and levelsof NF-κB p50 and p65 are determined according to the recommendations ofthe manufacturer. All experiments are performed in triplicate.

Identification and Quantitation of sTREM-1 Release

Primary monocytes suspensions are cultured as described above. The cellsare treated with E. coli LPS (O111:B4, 1 ug/mL) for 24 hours at 37° C.Cell-conditioned medium is submitted to Western-blotting using ananti-TREM-1 monoclonal antibody (R&D Systems) in order to confirm thepresence of 27 kDa material recognized by anti-TREM-1. Soluble TREM-1levels are measured by assessing the optical intensity of bands onimmunodots by means of a reflectance scanner and the Quantity OneQuantitation Software (Bio-Rad, Cergy, France). Soluble TREM-1concentration from each sample is determined by comparing the opticaldensities of the samples with reference to standard curves generatedwith purified TREM-1. All measurements are performed in triplicate. Thesensitivity of this technique allows the detection of sTREM-1 levels aslow as 5 ug/mL.

CLP Polymicrobial Sepsis Model

Male Balb/C mice (7 to 9 weeks, 20 to 23 g) are anaesthetized by i.p.administration of ketamine and xylazine in 0.2 mL sterile pyrogen-freesaline. The caecum is exposed through a 1.0 cm abdominal midlineincision and subjected to a ligation of the distal half followed by twopunctures with a G21 needle. A small amount of stool is expelled fromthe punctures to ensure patency. The caecum is replaced into theperitoneal cavity and the abdominal incision closes in two layers. Aftersurgery all mice are injected s.c. with 0.5 ml of physiologic salinesolution for fluid resuscitation and s.c. every 12 h with 1.25 mg (i.e.50 ug/g) of imipenem. The animals are randomly grouped and treated withnormal saline (n=14), control peptide (n=14; 100 ug) or TREM-1 CP (100ug) in a single injection at H0 (n=18), H+4 (n=18) or H+24 (n=18). Thelast group of mice (n=18) is treated with repeated injections of CP (100ug) at H+4, H+8 and H+24. All treatments are diluted into 500 ul ofnormal saline and administered i.p. To determine the effect of variousdoses of CP, mice (n=15 per group) are treated with a single injectionof normal saline or 10 ug, 20 ug, 50 ug, 100 ug or 200 ug of CP at H0after the CLP and monitored for survival. Five additional animals pergroup are killed under anaesthesia 24 hours after CLP for thedetermination of bacterial count and cytokines levels. Peritoneal lavagefluid is obtained using 2 mL RPMI 1640 (Life Technologies) and blood iscollected by cardiac puncture. Concentrations of TNF-alpha and IL-1betain the serum are determined by ELISA (BD Biosciences). For theassessment of bacterial counts, blood and peritoneal lavage fluid areplated in serial log dilutions on tryptic soy supplemented with 5% sheepblood agar plates. After plating, tryptic soy agar plates were incubatedat 37° C. aerobically for 24 hours, and anaerobically for 48 hours.Results are expressed as CFU per mL of blood and CFU per mouse for theperitoneal lavage.

Statistical Analyses

The protection against LPS lethality by CP is assessed by comparison ofsurvival curves using the Log-Rank test. All statistical analyses arecompleted with Statview software (Abacus Concepts, Berkeley Calif.).

Example 10: Haemodynamic Studies in Septic Rats Treated with TREM-1Peptide Variants

The role of TREM-1 peptides in further models of septic shock, isinvestigated by performing LPS- and CLP-induced endotoxinemiaexperiments in rats. The experiments can be conducted analogously tothat described in US Pat Appl 20060246082, which is incorporated hereinby reference in its entirety.

Materials and Methods

LPS-Induced Endotoxinemia

Animals are randomly grouped (n=10-20) and treated with Escherichia coliLPS (O111:B4, Sigma-Aldrich, Lyon, France) i.p. in combination withTREM-1 CP or CP-A at various concentrations.

CLP Polymicrobial Sepsis Model

This procedure has been described in details elsewhere (Mansart et al.Shock 2003; 19:38-44). Briefly, rats (n=6-10 per group) are anesthetizedby i.p. administration of ketamine (150 mg/kg). The caecum is exposedthrough a 3.0-cm abdominal midline incision and subjected to a ligationof the distal half followed by two punctures with a G21 needle. A smallamount of stool is expelled from the punctures to ensure potency. Thecaecum is replaced into the peritoneal cavity and the abdominal incisionclosed in two layers. After surgery, all rats are injected s.c. with 50mL/kg of normal saline solution for fluid resuscitation. TREM-1 CP orCP-A peptides are then administered at various concentrations.

Haemodynamic Measurements in Rats

Immediately after LPS administration as well as 16 hours after CLP,arterial BP (systolic, diastolic, and mean), heart rate, abdominalaortic blood flow, and mesenteric blood flow are recorded using aprocedure described elsewhere (Mansart et al. Shock 2003; 19:38-44).Briefly, the left carotid artery and the left jugular vein arecannulated with PE-50 tubing. Arterial BP is continuously monitored by apressure transducer and an amplifier-recorder system (IOX EMKATechnologies, Paris, France). Perivascular probes (Transonic Systems,Ithaca, N.Y.) are wrapped up the upper abdominal aorta and mesentericartery, allowed to monitor their respective flows by means of aflowmeter (Transonic Systems). After the last measurement (4^(th) hourafter LPS and 24^(th) hour after CLP), animals are sacrificed by anoverdose of sodium thiopental i.v.

Biological Measurements

Blood is sequentially withdrawn from the left carotid artery. Arteriallactate concentrations and blood gases analyses are performed on anautomatic blood gas analyser (ABL 735, Radiometer, Copenhagen, Denmark).Concentrations of TNF-alpha and IL-1beta in the plasma are determined byan ELISA test (Biosource, Nivelles, Belgium) according to therecommendations of the manufacturer. Plasmatic concentrations ofnitrates/nitrites are measured using the Griess reaction (R&D Systems,Abingdon, UK).

Statistical Analyses

Between-group comparisons are performed using Student't tests. Allstatistical analyses are completed with Statview software (AbacusConcepts, Calif.).

Example 11: Effect of TREM-1 Peptide Variants on Production of Cytokinesand Chemokines, Degranulation, and Expression of Cell Surface ActivationMarkers

These assays (as described in US Pat Appl 20090081199, and incorporatedherein by reference in its entirety) can be performed to demonstratethat the TREM-1 peptide variants are effective in inhibitingTREM-1-mediated cell activation.

Stimulation of TREM-1

To examine whether TREM-1 can trigger acute inflammatory responses,purified monocytes or neutrophils are stimulated for 24 h in 96-wellflat-bottom plates coated with F(ab′)₂ goat anti-mouse IgG (5 ug/ml)followed by either 21C7, 1 F11 (anti-MHC class I), or 1B7.11 (anti-2,4,6TNP) mAbs. Cells are plated at a concentration of 5×10⁴ cells/well inthe presence or absence of LPS (1 ug/ml). Supernatants are collected andtested for production of IL-6, IL-8, IL-10, IL-12p75, monocytechemoattractant protein-1 (MCP-1), TNF-alpha, and myeloperoxidase (MPO)by ELISA (PharMingen, San Diego, Calif.). To measure the expression ofcell surface markers, monocytes and neutrophils are stimulated asdescribed above and, after 48 hours, are stained with PE- orFITC-conjugated anti-CD11b, anti-CD11c, anti-CD18, anti-CD29, anti-CD32,anti-CD40, anti-CD49d, anti-CD49e, anti-CD54, anti-CD80, anti-CD83, oranti-CD86 (all from Immunotech, Marseille, France) and analyzed by FACS.To demonstrate TREM-1 inhibitory activity of TREM-1 peptide variants,either a TREM-1 peptide variant (e.g., the TREM-1 CP peptide, GFLSKSLVF)(SEQ ID NO: 16), or a sequence-scrambled control peptide (e.g.,LFGFLVSSK) (SEQ ID NO: 102), or a TREM-1 CP-A control peptide(GFLSASLVF) (SEQ IDNO: 88) are added to cells at various concentrationsbefore stimulation.

Measurement of Cytosolic Ca²⁺ and Tyrosine-Phosphorylated Proteins

Determination of intracellular Ca²⁺ mobilization can be done accordingto the previous reports (Nakajima et al. J Immunol 1999; 162:5-8).Briefly, monocytes or monocyte-derived DCs are loaded with Indo-1 AM dye(Sigma) for 30 min at 37° C., washed 3 times and resuspended in RPMI-10mM HEPES/10% FCS. Cytoplasmic Ca²⁺ levels are monitored in individualcells by measuring 405/525 spectral emission ratio of loaded Indo-1 dyeby flow cytometry (Nakajima et al. J Immunol 1999; 162:5-8; Yamashita etal. J Immunol 1998; 161:4042-7). After obtaining the baseline for atleast 30 seconds, for TREM-1 stimulation, anti-TREM-1 mAb or anti-MHCclass I (isotype-matched control mAb) and a cross-linking Ab (goatanti-mouse IgG) are added to the monocytes, and analysis is allowed tocontinue. For TREM-2 stimulation, either 29E3^(Biotin) (IgG1, kappa orFab) or 21C7^(Biotin) (IgG1, kappa or Fab) is added to a finalconcentration of 1 μg/ml and analysis is continued up to 512 sec. Insome experiments, ExtraAvidine (Sigma) is added as cross-linker togetherwith the biotinylated primary antibodies or antibody fragments. Todemonstrate TREM-1 inhibitory activity of TREM-1 peptide variants,either a TREM-1 peptide variant (e.g., the TREM-1 CP peptide, GFLSKSLVF)(SEQ ID NO: 16), or a sequence-scrambled control peptide (e.g.,LFGFLVSSK) (SEQ ID NO: 102), or a TREM-1 CP-A control peptide(GFLSASLVF) (SEQ ID NO: 88) are added to cells at various concentrationsbefore TREM-1 stimulation.

Determination of protein tyrosine phosphorylation, mitogen activatedprotein kinase activation, phospholipase C-gamma(PLC-gamma)phosphorylation, and immunoprecipitations can be performed as previouslydescribed (Dietrich et al. J Immunol 2000; 164:9-12). Briefly, monocytesare incubated at 37° C. with 27Cl mAb (anti-TREM-1) or control IgG1(anti-MHC class I) mAbs in the presence of a cross-linking Ab for theindicated time periods. After stimulation, an aliquot of the cells islysed and subjected to anti-phosphotyrosine blotting using PY-20(Transduction Laboratories, Lexington, Ky.). Another aliquot ofstimulated monocytes or monocyte-derived DCs is examined by Western blotanalysis using anti-phospho-extracellular signal-regulated kinase ½(P-ERK1/2) and anti-ERK1/2 mAbs. Tyrosine phosphorylated proteins areprecipitated from the stimulated monocyte lysates and immunoblotted withanti-PLC-gamma or anti-Hck Abs. An anti-Hck blotting is performed as aloading control because phosphorylation of Hck is similar in bothstimulated and unstimulated monocytes. To demonstrate TREM-1 inhibitoryactivity of TREM-1 peptide variants, either a TREM-1 peptide variant(e.g., the TREM-1 CP peptide, GFLSKSLVF) (SEQ ID NO: 16), or asequence-scrambled control peptide (e.g., LFGFLVSSK) (SEQ ID NO: 102),or a TREM-1 CP-A control peptide (GFLSASLVF) (SEQ ID NO: 88) are addedto cells at various concentrations before stimulation.

Example 12: Attenuation of Intestinal Inflammation in Animal Models ofColitis with TREM-1 Peptide Variants

In order to demonstrate that the TREM-1 peptide variants are effectivein inhibiting TREM-1-mediated cell activation in animal models ofcolitis, the experiments can be conducted analogously to that describedin Schenk et al. J Clin Invest 2007; 117:3097-106, which is incorporatedherein by reference in its entirety.

Methods

Mice

C57BL/6 mice, purchased from Harlan, and C57BL/6 RAG2−/− mice, bred in aspecific pathogen-free (SPF) animal facility, are used at 8-12 weeks ofage. All experimental mice are kept in micro-isolator cages in laminarflows under SPF conditions.

Mousemodels of Colitis

For experiments involving the adoptive T cell transfer model, colitis isinduced in C57BL/6 RAG2−/− mice by adoptive transfer of sortedCD4+CD45RBhigh T cells. Briefly, CD4+ T cells are isolated fromsplenocytes from C57BL/6 mice, and after osmotic lysis of erythrocytes,CD4+ T cells are enriched by a negative MACS procedure for CD8alpha andB220 (purified, biotinylated, hybridoma supernatant) usingavidin-labeled magnetic beads (Miltenyi Biotec). Subsequently, the CD4+T cell-enriched fraction is stained and FACS sorted for CD4+ (RM4-5; BDBiosciences—Pharmingen), CD45RBhi (16 A BD Biosciences—Pharmingen), andCD25-(PC61; eBioscience) naive T cells. Each C57BL/6 RAG2−/− mouse isinjected i.p. with 1×105 syngeneic CD4+CD45RBhighCD25-T cells. Coliticmice are sacrificed and analyzed on day 14 after adoptive transfer.

For experiments involving the dextran sodium sulfate (DSS) colitismodel, C57BL/6 mice are given autoclaved tapwater containing 3% DSS (DSSsalt, reagent grade, molwt: 36-50 kDa; MP Biomedicals) ad libitum over a5-dayperiod. The consumption of 3% DSS is measured. DSS is replacedthereafter by normal drinking water for another 4 days. Mice areeuthanized and analyzed at the end of the 9-day experimental period.

TREM-1 Peptide Treatment

Upon colitis induction, either starting on day or after onset of colitison day 3 (as indicated), mice are treated with either a TREM-1 peptidevariant (e.g., the TREM-1 CP peptide, GFLSKSLVF) (SEQ ID NO: 16), or asequence-scrambled control peptide (e.g., LFGFLVSSK) (SEQ ID NO: 102),or a TREM-1 CP-A control peptide (GFLSASLVF) (SEQ ID NO: 88), aspreviously described for septic shock models by Gibot and coworkers(Gibot S., et al. J Exp Med 2004; 200:1419-26). The peptides arechemically synthesized as described herein. Mice are treated once dailywith 200 ug peptide, injected i.p. in 200 ul saline.

Colitis Scoring

At the end of the experiments, the colon length is measured from the endof the cecum to the anus. Fecal samples are tested for occult bloodusing hemo FEC (Roche) tests (score 0, negative test; 1, positive testand no rectal bleeding; 2, positive test together with visible rectalbleeding). The colon is divided into 2 parts. From each mouse, identicalsegments from the distal and proximal colon are taken for protein andRNA isolation and histology, and frozen tissue blocks are prepared forsubsequent analysis. Histological scoring of paraffin-embeddedH&E-stained colonic sections is performed in a blinded fashionindependently by 2 pathologists. To assess the histopathologicalalterations in the distal colon, a scoring system is established usingthe following parameters: (a) mucin depletion/loss of goblet cells(score from 0 to 3); (b) crypt abscesses (score from 0 to 3); (c)epithelialerosion (score from 0 to 1); (d) hyperemia (score from 0 to2); (e) cellular infiltration (score from 0 to 3); and (f) thickness ofcolonic mucosa (score from 1 to 3). These individual histology scoresare added to obtain the final histopathology score for each colon (0, noalterations; 15, most severe signs of colitis).

RNA Isolation and RT-PCR

RNA is isolated from intestinal tissue samples preserved in RNAlater(QIAGEN), using the RNAeasy Mini Kit (QIAGEN). RT-PCR is performed with400 ng RNA each, using the TaqMan Gold RT-PCR Kit (Applied Biosystems).Primers are designed as follows: mouse TREM-1, forward5′-GAGCTTGAAGGATGAGGAAGGC-3′ (SEQ ID NO: 89) and reverse5′-CAGAGTCTGTCACTTGAAGGTCAGTC-3′ (SEQ ID NO: 90); mouse TNF, forward5′-GTAGCCCACGTCGTAGCAAA-3′ (SEQ ID NO: 91) and reverse5′-ACGGCAGAGAGGAGGTTGAC-3′ (SEQ ID NO: 92); mouse beta-actin, forward5′-TGGAATCCTGTGGCATCCATGAAAC-3′ (SEQ ID NO: 93) and reverse5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (SEQ ID NO: 94); human TREM-1, forward5′-CTTGGTGGTGACCAAGGGTTTTTC-3′ (SEQ ID NO: 95) and reverse5′-ACACCGGAACCCTGATGATATCTGTC-3′ (SEQ ID NO: 96); human TNF, forward5′-GCCCATGTTGTAGCAAACCC-3′ (SEQ ID NO: 97) and reverse5′-TAGTCGGGCCGATTGATCTC-3′ (SEQ ID NO: 98); human GAPDH (SEQ ID NO: 99),forward 5′-TTCACCACCATGGAGAAGGC-3′ (SEQ ID NO: 100) and reverse5′-GGCATGGACTGTGGTCATGA-3′ (SEQ ID NO: 101). PCR products aresemiquantitatively analyzed on agarose gels.

Human TREM-1 and mouse TREM-1 and TNF expression is also assessed byreal-time PCR using the TREM-1 QuantiTect primer assay system andQuantiTect SYBR green PCR Kit (both from QIAGEN). GAPDH is used tonormalize TREM-1 and TNF expression levels. DNA is amplified on a 7500Real-Time PCR system (Applied Biosystems), and the increase in geneexpression is calculated using Sequence Detection System software(Applied Biosystems).

Western Blot Analysis

Protein samples are separated on a denaturing 12% acrylamide gel,followed by transfer to nitrocellulose filter and probing with theprimary antibody. Anti-TREM-1 (polyclonal goat IgG, 0.1 ug/ml; R&DSystems) or anti-tubulin (clone B-5-1-2, 1:5,000; Sigma-Aldrich) is usedas primary reagent. As secondary antibodies, HRP-labeled donkeyanti-goat Ig (1:2,000; The Binding Site) and goat anti-mouse Ig(1:4,000; Sigma-Aldrich) are used. Binding is detected bychemiluminescence using a Super Signal West Pico Kit (Pierce).

Statistics

The unpaired 2-tailed Student t test is used to compare groups; P valuesless than 0.05 are considered significant.

Example 13: Modulation of the TREM-1 Pathway by Means of TREM-1 PeptideVariants During Severe Hemorrhagic Shock in Rats

In order to demonstrate that the TREM-1 peptide variants are effectivein inhibiting TREM-1-mediated cell activation and preventing organdysfunction and improving survival in rats during severe hemorrhagicshock, the experiments can be conducted analogously to that described inGibot et al. Shock 2009; 32:633-7, which is incorporated herein byreference in its entirety.

Materials and Methods

Animals

Adult male Wistar rats (250-300 g) are purchased from Charles RiverLaboratories (Wilmington, Mass., USA). After 1 week of acclimatization,rats are fasted 12 h before the experiments and are allowed free accessto water. All the studies described in the succeeding sentences complywith the regulations concerning animal use and care published by theNational Institutes of Health.

TREM-1 Peptide Variants

As described herein, TREM-1 peptide variants (e.g., the TREM-1 CPpeptide, GFLSKSLVF) (SEQ ID NO: 16) are chemically synthesized. Ascrambled peptide containing the same amino acids but in a totallydifferent sequence order (e.g., LFGFLVSSK) (SEQ ID NO: 102) is similarlysynthesized and serves as control peptide. Alternatively, a TREM-1 CP-Apeptide (GFLSASLVF) (SEQ ID NO: 88) is chemically synthesized and servesas control peptide.

Hemorrhagic Shock Model

Hemorrhagic shock is induced by bleeding from a heparinized (10 UI/mL)carotid artery catheter. Briefly, the rats are anesthetized (50 mg/kgpentobarbital sodium, i.p.) and kept on a temperature-controlledsurgical board (37° C.). A tracheostomy is performed, and the animalsare ventilated supine (tidal volume, 7-8 mL/kg; rodent ventilator no.683; Harvard Apparatus, Holliston, Mass) with a fraction of inspiredoxygen of 0.3 and a respiratory rate of 60 breaths per minute.Anesthesia and respiratory support are maintained during the wholeexperiment. The left carotid artery and the left jugular vein arecannulated with PE-50 tubing. Arterial blood pressure is continuouslymonitored by a pressure transducer and an amplifier-recorder system (IOXEMKA Technologies, Paris, France). After a 30-min stabilization period,blood is drawn in 10 to 15 min via the carotid artery catheter until MAPreached 40 mmHg. Blood is kept at 37° C., and MAP is maintained between35 and 40 mm Hg during 60 min. Rats are then allocated randomly (n=10-12per group) to receive 0.1 mL of either saline (isotonic sodium chloridesolution), TREM-1 peptide variant, or control peptide (various amountsof peptides in 0.1 mL of saline) solution over 1 min via the jugularvein (H0). Shed blood and ringer lactate (volume=3× shed volume) arethen infused via the jugular vein in 60 min, and rats are observed for a4-h period before being killed by pentobarbital sodium overdose. Killingoccurs earlier if MAP decreased to less than 35 mm Hg.

Arterial Blood Gas, Lactate, and Cytokines

Arterial blood gas and lactate concentrations are determined hourly onan automatic blood gas analyzer (ABL 735; Radiometer, Copenhagen,Denmark). Concentrations of TNF-alpha and IL-6 and sTREM-1 in the plasmaare determined in triplicate by enzyme-linked immunosorbent assay(Biosources, Nivelles, Belgium; RnD Systems, Lille, France).

Bacterial Translocation

Rats are killed under anesthesia, and mesenteric lymph node (MLN)complex, spleen, and blood are aseptically removed 4 h after thebeginning of reperfusion (or earlier if MAP decreased <35 mm Hg).Homogenates of MLN and spleen and serial blood dilutions are plated andincubated overnight at 37° C. on Columbia blood agar plates (in carbondioxide and anaerobically) and Macconkey agar (in air). Visible coloniesare then counted.

Pulmonary Integrity

Additional groups of rats (n=4) are subjected to the same procedure butare also infused via the tail vein with fluorescein isothiocyanate(FITC)-albumin (5 mg/kg in 0.3 mL of phosphate-buffered saline) 2 hafter the beginning of reperfusion. Rats in these groups are killed 2 hlater with an overdose of sodium pentobarbital (200 mg/kg). Immediatelythereafter, the lungs are lavaged three times with 1 mL ofphosphate-buffered saline, and blood is collected by cardiac puncture.The bronchoalveolar lavage fluid (BALF) is pooled, and plasma iscollected. Fluorescein isothiocyanate-albumin concentrations in BALF andplasma are determined fluorometrically (excitation, 494 nm; emission,520 nm). The BALF-plasma fluorescence ratio is calculated and used as ameasure of damage to pulmonary alveolar endotheliallepithelial integrityas previously described (Yang et al. Crit Care Med 2004; 32:1453-9).

Statistical Analysis

Data are analyzed using ANOVA or ANOVA for repeated measures whenappropriate, followed by Newman-Keuls post hoc test. Survival curves arecompared using the log-rank test. A two-tailed value of P less than 0.05is deemed significant. All analyses are performed with GraphPad Prismsoftware (GraphPad, San Diego, Calif.).

Example 14: Modulation of the TREM-1 Pathway by Means of TREM-1 PeptideVariants in a Mouse Model of Human Non-Small Cell Lung CancerTumorigenesis

In order to demonstrate that the TREM-1 peptide variants are effectivein inhibiting TREM-1-mediated cell activation and reducing tumor growth,tumor vascularity, and spontaneous metastases in animal models of humannon-small cell lung cancer (NSCLC), the experiments can be conductedanalogously to those disclosed in US Pat Appl 20080193375 and describedelsewhere (Arenberg et al. J Clin Invest 1998; 102:465-72; Arenberg etal. J Clin Invest 1996; 97:2792-802; Arenberg et al. J Exp Med 1996;184:981-92; Phillips et al. Am J Respir Crit Care Med 2003;167:1676-86), which are incorporated herein by reference in itsentirety.

Materials and Methods

TREM-1 Peptide Variants

As described herein, TREM-1 peptide variants (e.g., the TREM-1 CPpeptide, GFLSKSLVF) (SEQ ID NO: 16) are chemically synthesized. Ascrambled peptide containing the same amino acids but in a totallydifferent sequence order (e.g., LFGFLVSSK) (SEQ ID NO: 102) is similarlysynthesized and serves as control peptide. Alternatively, a TREM-1 CP-Apeptide (GFLSASLVF) (SEQ ID NO: 88) is chemically synthesized and servesas control peptide.

Human NSCLC-SCID Mouse Chimeras

4-6-wk-old female CB17-SCID mice (Taconic Farms, Germantown, N.Y.) withserum Ig<1 ug/ml are injected subcutaneously with human NSCLC cells(1×10⁶ cells in 100 ul) into each flank. The animals are maintainedunder sterile conditions in laminar flow rooms and killed in groups ofsix. At time of death, anticoagulated (heparin 50 U/500 ul of blood)blood is collected and centrifuged. The plasma is stored at −70° C. forlater analysis. Tumors are dissected from the mice and measured with aThorpe caliper (Biomedical Research Instruments, Rockville, Md.). Aportion of the tumor is fixed in 4% paraformaldehyde for histologicanalysis and immunohistochemistry. H & E-stained sections are examinedunder 400× magnification to quantify infiltrating neutrophils. 10 fieldsare examined in each of nine tumor sections from both TREM-1 peptidevariant- and control peptide-treated groups. Results are expressed asthe number of cells per high power field (HPF; 400×). The other portionof the tumor is snap frozen for subsequent homogenization and sonicationin antiprotease buffer followed by filtration through 0.45-um filters(Acrodiscs, Gelman Sciences, Ann Arbor, Mich.). The filtrate is storedat 70° C. for later analysis. All tumor homogenates are standardized fortotal protein prior to lyophilization (SpeedVac, Savant) and used in thecorneal micropocket model of neovascularization. The right lung isinflated with 4% paraformaldehyde, and prepared for histopathologicanalysis, or processed for FACS analysis (CD49b) of human cellpopulations (A549 cells). In the TREM-1 inhibition studies, SCID micereceive intraperitoneal (i.p.) injections of 500 ul of either TREM-1peptide variant, or control peptide, or no treatment, every 48 h for 6wk, starting at the time of cell inoculation. Tumor specimens from thesemice are processed as described above.

Corneal Micropocket Model of Angiogenesis.

In vivo angiogenic activity of the tumors is assayed in the avascularcornea of Long Evans rat eyes, as previously described (Koch et al.Arthritis Rheum 1989; 29:471-9). Briefly, equal volumes of lyophilizedtumor specimens normalized to total protein, are combined with sterileHydron (Interferon Sciences Inc.) casting solution. 5-ul aliquots arepipetted onto the flat surface of an inverted sterile polypropylenespecimen container, and polymerized overnight in a laminar flow hoodunder UV light. Prior to implantation, pellets are rehydrated withnormal saline. Animals are given i.p. ketamine (150 mg/kg) and atropine(250 ug/kg) for anesthesia. Rat corneas are anesthetized with 0.5%proparacaine hydrochloride ophthalmic solution followed by implantationof the Hydron pellet into an intracorneal pocket (1-2 mm from thelimbus). 6 d after implantation, animals receive heparin (1000 U) of andketamine (150 mg/Kg) i.p., followed by a 10 ml perfusion of colloidalcarbon via the left ventricle. Corneas are harvested and photographed.Positive neovascularization responses were defined as sustaineddirectional in growth of capillary sprouts and hairpin loops towards theimplant are observed. Negative responses are defined as either no growthor only an occasional sprout or hairpin loop displaying no evidence ofsustained growth.

Quantitation of Vessel Density

Quantitation of vessel density is performed using a modification of thepreviously described method (Weidner N. Am J Pathol 1995; 147:9-19).Briefly, tissue sections are dewaxed with xylene and rehydrated throughgraded concentrations of ethanol. Slides are blocked with normal rabbitserum (BioGenex, San Ramon, California), and overlaid with 1:500dilution of either control (goat) or goat anti-Factor VIII-relatedantigen antibodies. Slides are then rinsed and overlaid with secondarybiotinylated rabbit anti-goat IgG (1:35) and incubated for 60 min. Afterwashing twice with Tris-buffered saline, slides are overlaid with a 1:35dilution of alkaline phosphatase conjugated to streptavidin (BioGenex),and incubated for 60 min. Fast Red (BioGenex) reagent was used forchromogenic localization of Factor VIII antigen. After optimal colordevelopment, sections are immersed in sterile water, counterstained withMayer's hematoxylin, and cover slipped using an aqueous mountingsolution. A549 tumor specimens from TREM-1 peptide variant- and controlpeptide-treated SCID mice are examined in a blinded fashion for thepresence of Factor VIII immunolocalization. Sections are first scannedat low magnification (40×) to identify vascular “hot spots.” Areas ofgreatest vessel density are then examined under higher magnification(400×) and counted. Any distinct area of positive staining for FactorVIII is counted as a single vessel. Results are expressed as the meannumber of vessels±SEM per high power field (HPF; 400×). A total of 30HPFs are examined and counted from three tumors of each of the treatmentgroups.

FACS analysis for human CD49b (A549 lung metastases). Before removal,lungs from human NSCLC tumor bearing animals are perfused with normalsaline, and dissected free of the thoracic cavity. The right lung isminced, and incubated for 1 h in digestion media (RPMI with 0.02%collagenase type IV, and 0.1 mg of bovine pancreas grade II DNasel).Cells are further separated by repeatedly aspirating the cell suspensionthrough a 20-ml syringe. Cells are then pelleted at 600 g for 10 min,resuspended in sterile water for 30 s to lyse remaining RBCs, washed in1× PBS, and resuspended in complete media with 5% FCS. Cells arecounted, transferred at a concentration of 5×10⁶ cells/ml to fluorescentantibody buffer (1% FA buffer [Difco No. 2314-15], 1% FCS, and 0.1%azide), and maintained at 4° C. for the remainder of the stainingprocedure. 100 ul of cells are labeled with FITC-conjugated ratanti-human CD49b (1 ug, Pharmingen, San Diego, Calif.). This antibodyrecognizes the alpha-2 portion of the beta-1-integrin, VLA-2, a markerpreviously found to be expressed by A549 and Calu 1 cells.FITC-conjugated rat IgG is used as a control antibody. Unbound antibodyis washed with FA buffer and the cell suspension is analyzed with FACS(Becton Dickinson). The data are expressed as the percentage of cellsstaining positively with anti-human CD49b.

Statistical Analysis

The studies involve a minimum of six human NSCLC/SCID mouse chimeras ateach time point or for each manipulation. Groups of data are evaluatedby analysis of variance to indicate groups with significant differences.Data that appear statistically significant are compared by Student's ttest for comparing the means of multiple groups, and are consideredsignificant if p values are less than 0.05. Results were presented asmeans±SEM. Data are analyzed using Statview II statistical softwarepackage (Abacus Concepts, Inc.).

INCORPORATION BY REFERENCE

All of the patents and publications cited herein are hereby incorporatedby reference. Each of the applications and patents cited in this text,as well as each document or reference cited in each of the applicationsand patents (including during the prosecution of each issued patent;“application cited documents”), and each of the PCT and foreignapplications or patents corresponding to and/or paragraphing priorityfrom any of these applications and patents, and each of the documentscited or referenced in each of the application cited documents, arehereby expressly incorporated herein by reference. More generally,documents or references are cited in this text, either in a ReferenceList, or in the text itself; and, each of these documents or references(“herein-cited references”), as well as each document or reference citedin each of the herein-cited references (including any manufacturer'sspecifications, instructions, etc.), is hereby expressly incorporatedherein by reference.

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A composition comprising a peptide having aformula consisting of R1-G-X1-X2-L-S-X3-X4-L-V-X5-X6-X7-X8-X9-X10-R2(SEQ ID NO: 103), wherein: R1 is absent or is selected from the groupconsisting of N-terminal sugar conjugate and N-terminal lipid conjugate;X1 is selected from the group consisting of Gly, Cys, Arg, Lys and His;X2 is selected from the group consisting of Leu, Phe, and Ile; X3 isselected from the group consisting of Arg, Lys and His; X4 is selectedfrom the group consisting of Ser and Thr; X5 is selected from the groupconsisting of Phe, Arg, Lys and His; X6 is absent or is selected fromthe group consisting of Ser, Leu and Ile; X7 is absent or is selectedfrom the group consisting of Val, Ile, Leu and Gly; X8 is absent or isselected from the group consisting of Leu, Phe, and Ala; X9 is absent oris selected from the group consisting of Leu, Phe, and Ala; X10 isabsent or is selected from the group consisting of Leu, Phe, and Ala;and R2 is absent or is C-terminal lipid conjugate.
 2. The composition ofclaim 1, wherein said N-terminal sugar conjugate is 1-amino-glucosesuccinate.
 3. The composition of claim 1, wherein said N-terminal lipidconjugate is selected from the group consisting of 2-aminododecanoateand myristoylate conjugates.
 4. The composition of claim 1, wherein saidC-terminal lipid conjugate is selected from the group consisting ofGly-Tris-monopalmitate, Gly-Tris-dipalmitate and Gly-Tris-tripalmitateconjugates.
 5. The composition of claim 1, wherein the peptide isattached to a carrier molecule.
 6. The composition of claim 1, whereinthe peptide is conjugated at a free amine group with a polyalkyleneglycol.
 7. The composition of claim 6, wherein the said polyalkyleneglycol is polyethylene glycol.
 8. The composition of claim 1, whereinone or more amino acid is a D-amino acid.
 9. The composition of claim 1,wherein said amino acid sequence further comprises at least one aminoacid selected from the group consisting of an L-amino acid and a D-aminoacid.
 10. The composition of claim 1, wherein said peptide is a cyclicpeptide.
 11. The composition of claim 1, wherein said peptide is acyclic dimer peptide.
 12. The composition of claim 1, wherein saidpeptide is a dimer peptide.
 13. A method comprising: a) providing i) apatient having at least one symptom of a myeloid cell-mediatedphathology; and ii) a composition consisting ofR1-G-X1-X2-L-S-X3-X4-L-V-X5-X6-X7-X8-X9-X10-R2 (SEQ ID NO: 103),wherein: R1 is absent or is selected from the group consisting ofN-terminal sugar conjugate and N-terminal lipid conjugate; X1 isselected from the group consisting of Gly, Cys, Arg, Lys and His; X2 isselected from the group consisting of Leu, Phe, and Ile; X3 is selectedfrom the group consisting of Arg, Lys and His; X4 is selected from thegroup consisting of Ser and Thr; X5 is selected from the groupconsisting of Phe, Arg, Lys and His; X6 is absent or is selected fromthe group consisting of Ser, Leu and Ile; X7 is absent or is selectedfrom the group consisting of Val, Ile, Leu and Gly; X8 is absent or isselected from the group consisting of Leu, Phe, and Ala; X9 is absent oris selected from the group consisting of Leu, Phe, and Ala; X10 isabsent or is selected from the group consisting of Leu, Phe, and Ala;and R2 is absent or is C-terminal lipid conjugate, and; b) administeringsaid composition to said patient under conditions such that said atleast one symptom is reduced.
 14. The method of claim 13, wherein saidmyeloid cell-mediated pathology is selected from the group comprisingsepsis, cancer, inflammatory bowel disease, acute mesenteric ischemia,hemorrhagic shock, arthritis, ankylosing spondylitis, fibromyalgia,lupus, sclerodema, polymyositis, dermatomyositis, polymyalgiarheumatica, bursitis, tendinitis, vasculitis, carpal tunnel syndrome,complex regional pain syndrome, juvenile arthritis, Lyme disease,systemic lupus erythematosus, Kawasaki disease, chronic fatiguesyndrome, myositis, tissue rejection and organ rejection.
 15. Abiocompatible medical device comprising a coating, wherein said coatingconsisting of R1-G-X1-X2-L-S-X3-X4-L-V-X5-X6-X7-X8-X9-X10-R2 (SEQ ID NO:103), wherein: R1 is absent or is selected from the group consisting ofN-terminal sugar conjugate and N-terminal lipid conjugate; X1 isselected from the group consisting of Gly, Cys, Arg, Lys and His; X2 isselected from the group consisting of Leu, Phe, and Ile; X3 is selectedfrom the group consisting of Arg, Lys and His; X4 is selected from thegroup consisting of Ser and Thr; X5 is selected from the groupconsisting of Phe, Arg, Lys and His; X6 is absent or is selected fromthe group consisting of Ser, Len and Ile; X7 is absent or is selectedfrom the group consisting of Val, Ile, Leu and Gly; X8 is absent or isselected from the group consisting of Leu, Phe, and Ala; X9 is absent oris selected from the group consisting of Leu, Phe, and Ala; X10 isabsent or is selected from the group consisting of Leu, Phe, and Ala;and R2 is absent or is C-terminal lipid conjugate.
 16. The device ofclaim 15, wherein said device is selected from the group consisting ofstents, grafts, catheters, endoscopes, atrial/venous fistulas, andcannulae.
 17. The device of claim 15, wherein the said coating furthercomprises a polymer.
 18. The device of claim 17, wherein said polymer isselected from the group consisting of phosphorylcholine, polyvinylpyrrolidone, poly(acrylic acid), poly(vinyl acetamide), poly(propyleneglycol), poly(ethylene co-vinyl acetate), poly(n-butyl methacrylate) andpoly(styrene-b-isobutylene-b-styrene).